Human Interferon Molecules and Their Uses

ABSTRACT

Modified human interferon polypeptides and uses thereof are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent application60/687,173 filed Jun. 3, 2005 entitled “Improved Human InterferonMolecules and Their Uses” and U.S. provisional patent application60/753,375 filed Dec. 21, 2005 entitled “Improved Human InterferonMolecules and Their Uses”, the specifications of which are incorporatedherein in their entirety.

FIELD OF THE INVENTION

This invention relates to interferon polypeptides modified with at leastone non-naturally encoded amino acid.

BACKGROUND OF THE INVENTION

The growth hormone (GH) supergene family (Bazan, F. Immunology Today 11:350-354 (1990); Mott, H. R. and Campbell, I. D. Current Opinion inStructural Biology 5: 114-121 (1995); Silvennoinen, O. and Ihle, J. N.(1996) SIGNALING BY THE HEMATOPOIETIC CYTOKINE RECEPTORS) represents aset of proteins with similar structural characteristics. Each member ofthis family of proteins comprises a four helical bundle. While there arestill more members of the family yet to be identified, some members ofthe family include the following: growth hormone, prolactin, placentallactogen, erythropoietin (EPO), thrombopoietin (TPO), interleukin-2(IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35subunit), IL-13, IL-15, oncostatin M, ciliary neurotrophic factor,leukemia inhibitory factor, alpha interferon, beta interferon, gammainterferon, omega interferon, tau interferon, epsilon interferon,granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophagecolony stimulating factor (GM-CSF), macrophage colony stimulating factor(M-CSF) and cardiotrophin-1 (CT-1) (“the GH supergene family”). Membersof the GH supergene family have similar secondary and tertiarystructures, despite the fact that they generally have limited amino acidor DNA sequence identity. The shared structural features allow newmembers of the gene family to be readily identified. The generalstructure of IFNα-2 is shown in FIG. 1.

Interferons are relatively small, single-chain glycoproteins released bycells invaded by viruses or exposed to certain other substances.Interferons are presently grouped into three major classes,designated: 1) leukocyte interferon (interferon-alpha, α-interferon,IFN-α), 2) fibroblast interferon (interferon-beta, β-interferon, IFN-β),and 3) immune interferon (interferon-gamma, γ-interferon, IFN-γ). Inresponse to viral infection, lymphocytes synthesize primarilyα-interferon (with omega interferon, IFN-ω), while infection offibroblasts usually induces production of β-interferon. IFNα and IFNβshare about 20-30 percent amino acid sequence homology. The gene forhuman IFN-β lacks introns, and encodes a protein possessing 29% aminoacid sequence identity with human IFN-α, suggesting that IFN-α and IFN-βgenes have evolved from a common ancestor (Taniguchi et al., Nature 285547-549 (1980)). By contrast, IFN-γ is synthesized by lymphocytes inresponse to mitogens. IFNα, IFN β and IFNω are known to induce MHC ClassI antigen expression and are referred to as type I interferons, whileIFNγ induces MHC Class II antigen expression, and is referred to as typeII interferon. Pestka et al. in Annu. Rev. Immunol. (2004) 22:929-79,which is incorporated by reference herein in its entirety, describesclass 2 α-helical cytokines including interferons (IFN-α, -β, -ε, -κ,-ω, -δ, -τ, and -γ) as well as interferon-like molecules such aslimitin, IL-28A, IL-28B, and IL-29 as well as the ligands, receptors,and signal transduction pathways employed by these molecules. Theinterferons have different species and many allelic variants. Inadditional, interferons with novel activities and mutant sequences havebeen isolated from cells from patients with various diseases.

A large number of distinct genes encoding different species of IFNα havebeen identified. Alpha interferons fall into two major classes, I andII, each containing a plurality of discrete proteins (Baron et al.,Critical Reviews in Biotechnology 10, 179-190 (1990); Nagata et al.,Nature 287, 401-408 (1980); Nagata et al., Nature 284, 316-320 (1980);Streuli et al., Science 209, 1343-1347 (1980); Goeddel et al., Nature290, 20-26 (1981); Lawn et al., Science 212, 1159-1162 (1981); Ullrichet al., J. Mol. Biol. 156, 467-486 (1982); Weissmann et al., Phil.Trans. R. Soc. Lond. B299, 7-28 (1982); Lund et al., Proc. Natl. Acad.Sci. 81, 2435-2439 (1984); Capon et al., Mol. Cell. Biol. 5, 768(1985)). The various IFN-αspecies include IFN-αA (IFN-α2), IFN-αB,IFN-αC, IFN-αCl, IFN-αD (IFN-α1), IFN-αE, IFN-αF, IFN-αG, IFN-αH,IFN-αI, IFN-αJ1, IFN-αJ2, IFN-αK, IFN-αL, IFN-α4B, IFN-α5, IFN-α6,IFN-α74, IFN-α76 IFN-α4a), IFN-α88, and alleles thereof. Trotta et al.in “Approval Standards for Alfa Interferon Subtypes” Drug InformationJournal 34:1231-1246 (2000), which is incorporated by reference hereinin its entirety, describe the members of the human IFN α gene family andproteins and the biological activities of this family including theimmunomodulatory, antiproliferative, anti-viral and anti-microbialactivities. The interferon proteins mentioned by Trotta et al. includeIFN α₁ (from IFN_(A1) gene), IFN α_(D), IFN α₂ (IFN α_(2b)), IFN α_(A)(IFN α_(2a)), IFNα_(2c), IFN α_(4a) (IFN α₇₆), IFN α_(4b), IFN α₅, IFNα_(G), IFN α₆₁, IFN α₆, IFN α_(κ), IFN α₅₄, IFN α₇, IFN α_(J), IFNα_(J1), IFN α₈, IFN α_(B2), IFN α_(B), IFN α_(C), ΨIFN α₁₀, ΨIFN α_(L),IFN α_(6L), IFN α₁₃, IFN α₁₄, IFN α_(H), IFN α_(H1), IFN α₁₆, IFNα_(WA), IFN α_(O), IFN α₁₇, IFN α₁ (from IFN_(A17) gene), IFN α₈₈, IFNα₁ (from IFN_(A21) gene), IFN α_(F), and ΨIFN α_(E). Trotta et al. alsodiscuss the production, characterization, quality assurance, biologicalactivity, and clinical safety and efficacy issues that relate torecombinant versions of proteins in this family. Release tests andphysicochemical characterization tests are also discussed. IFN α₂₁, IFNα₄, IFN α₁₀, and IFN α₃ are other interferon proteins that have beenpreviously described.

Interferons were originally derived from naturally occurring sources,such as buffy coat leukocytes and fibroblast cells, optionally usinginducing agents to increase interferon production. Interferons have alsobeen produced by recombinant DNA technology.

The cloning and expression of recombinant IFNαA (IFNαA, also known asIFNα2) was described by Goeddel et al., Nature 287, 411 (1980). Theamino acid sequences of IFNαA, B, C, D, F, G, H, K and L, along with theencoding nucleotide sequences, are described by Pestka in Archiv.Biochem. Biophys. 221, 1 (1983). The cloning and expression of matureIFNβ is described by Goeddel et al., Nucleic Acids Res. 8, 4057 (1980).The cloning and expression of mature IFNγ are described by Gray et al.,Nature 295, 503 (1982). IFNω has been described by Capon et al., Mol.Cell. Biol. 5, 768 (1985). IFNτ has been identified and disclosed byWhaley et al., J. Biol. Chem. 269, 10864-8 (1994).

Interferons have a variety of biological activities, includinganti-viral, immunoregulatory and anti-proliferative properties, and havebeen utilized as therapeutic agents for treatment of diseases such ascancer, and various viral diseases. As a class, interferon-α's have beenshown to inhibit various types of cellular proliferation, and areespecially useful for the treatment of a variety of cellularproliferation disorders frequently associated with cancer, particularlyhematologic malignancies such as leukemias. These proteins have shownanti-proliferative activity against multiple myeloma, chroniclymphocytic leukemia, low-grade lymphoma, Kaposi's sarcoma, chronicmyelogenous leukemia, renal-cell carcinoma, urinary bladder tumors andovarian cancers (Bonnem, E. M. et al. (1984) J. Biol. Response Modifiers3:580; Oldham, R. K. (11985) Hospital Practice 20:71).

In addition, interferon-α may have important neuroregulatory functionsin the CNS. Structural and functional similarities have been shownbetween IFNα and endorphins. It has been reported that the IFNα moleculecontains distinct domains that mediate immune and opioid-like effectsand that the μ opioid receptor may be involved in the analgesic effectof IFNα. Analgesic domains of the tertiary structure of interferon-αhave been described which locate around the 122^(nd) Tyr residue of themolecule and includes the Phe residues 36, 38, and 123 (Wang et al. J.Neuroimmunol. (2000) 108:64-67 and Wang et al. NeuroReport (2001)12(4):857-859, which are incorporated by reference herein).Specifically, Wang et al. found that an interferon-α mutant at residue36 (F36S) resulted in a complete loss of analgesic activity and areduction of anti-viral activity. Another IFN-α mutant (F38S) resultedin a complete loss of analgesic activity and almost a complete loss ofanti-viral activity. Other mutants of IFNα that have been studiedinclude F38L and Y129S. Wang et al. describe these two mutants instudies investigating fever induced by human IFNα, and found that thisside effect of IFNα therapy is mediated by IFNα's interaction withopioid receptor and a subsequent induction of prostaglandin E₂ (J. ofNeuroimmunology (2004) 156:107-112). Modulating the interaction betweenIFN and opioid receptors may be critical in the development of novel IFNtherapeutics to prevent side effects involving this family of receptors.Prostaglandins modulate CNS functions including but not limited to, thegeneration of fever, the sleep/wake cycle, and the perception of pain.They are produced by the enzymatic activity of cyclooxygenases COX-1 andCOX-2.

The administration of IFN-α may also result in a number ofneuropsychiatric side effects including depression (Wichers and Maes,Rev. Psychiat. Neurosci. (2004) 29(1):11-17). Wichers and Maes indicateserotonin (5-HT) brain neurotransmission and the induction of the enzymeIDO (indolamine 2,3-dioxygenase) are involved. Other hypotheses involvenitric oxide and soluble ICAM-1 induction by IFN. Modulating themechanisms by which IFN causes such side effects may be critical in thedevelopment of novel IFN therapeutics.

Specific examples of commercially available IFN products include IFNγ-1b(Actimmune®), IFNβ-1a (Avonex®, and Rebif®), IFNβ-1b (Betaseron®), IFNalfacon-1 (Infergen®), IFNα-2 (Intron A®), IFNα-2a (Roferon-A®),Peginterferon alfa-2a (PEGASYS®), and Peginterferon alfa-2b(PEG-Intron®). Some of the problems associated with the production ofPEGylated versions of IFN proteins are described in Wang et al. (2002)Adv. Drug Deliv. Rev. 54:547-570; and Pedder, S. C. Semin Liver Dis.2003; 23 Suppl 1:19-22. Wang et al. characterized positional isomers ofPEG-Intron®, and Pedder at al. compared PEGASYS® with PEG-Intron®describing the lability of the PEGylation chemistries used and effectsupon formulation. PEGASYS® is comprised of nine identifiable isoforms,which specific isoforms differing in anti-viral activity (Foser et al.,Pharmacogenomics J 2003; 3:312). Despite the number of IFN productscurrently available on the market, there is still an unmet need forinterferon therapeutics. In particular, interferon therapeutics thatmodulate one or more side effects found with current IFN therapeuticsare of interest.

Covalent attachment of the hydrophilic polymer poly(ethylene glycol),abbreviated PEG, is a method of increasing water solubility,bioavailability, increasing serum half-life, increasing therapeutichalf-life, modulating immunogenicity, modulating biological activity, orextending the circulation time of many biologically active molecules,including proteins, peptides, and particularly hydrophobic molecules.PEG has been used extensively in pharmaceuticals, on artificialimplants, and in other applications where biocompatibility, lack oftoxicity, and lack of immunogenicity are of importance. In order tomaximize the desired properties of PEG, the total molecular weight andhydration state of the PEG polymer or polymers attached to thebiologically active molecule must be sufficiently high to impart theadvantageous characteristics typically associated with PEG polymerattachment, such as increased water solubility and circulating halflife, while not adversely impacting the bioactivity of the parentmolecule.

PEG derivatives are frequently linked to biologically active moleculesthrough reactive chemical functionalities, such as lysine, cysteine andhistidine residues, the N-terminus and carbohydrate moieties. Proteinsand other molecules often have a limited number of reactive sitesavailable for polymer attachment. Often, the sites most suitable formodification via polymer attachment play a significant role in receptorbinding, and are necessary for retention of the biological activity ofthe molecule. As a result, indiscriminate attachment of polymer chainsto such reactive sites on a biologically active molecule often leads toa significant reduction or even total loss of biological activity of thepolymer-modified molecule. R. Clark et al., (1996), J. Biol. Chem.,271:21969-21977. To form conjugates having sufficient polymer molecularweight for imparting the desired advantages to a target molecule, priorart approaches have typically involved random attachment of numerouspolymer arms to the molecule, thereby increasing the risk of a reductionor even total loss in bioactivity of the parent molecule.

Reactive sites that form the loci for attachment of PEG derivatives toproteins are dictated by the protein's structure. Proteins, includingenzymes, are composed of various sequences of alpha-amino acids, whichhave the general structure H₂N—CHR—COOH. The alpha amino moiety (H₂N—)of one amino acid joins to the carboxyl moiety (—COOH) of an adjacentamino acid to form amide linkages, which can be represented as—(NH—CHR—CO)_(n)—, where the subscript “n” can equal hundreds orthousands. The fragment represented by R can contain reactive sites forprotein biological activity and for attachment of PEG derivatives.

For example, in the case of the amino acid lysine, there exists an —NH₂moiety in the epsilon position as well as in the alpha position. Theepsilon —NH₂ is free for reaction under conditions of basic pH. Much ofthe art in the field of protein derivatization with PEG has beendirected to developing PEG derivatives for attachment to the epsilon—NH₂ moiety of lysine residues present in proteins. “Polyethylene Glycoland Derivatives for Advanced PEGylation”, Nektar Molecular EngineeringCatalog, 2003, pp. 1-17. These PEG derivatives all have the commonlimitation, however, that they cannot be installed selectively among theoften numerous lysine residues present on the surfaces of proteins. Thiscan be a significant limitation in instances where a lysine residue isimportant to protein activity, existing in an enzyme active site forexample, or in cases where a lysine residue plays a role in mediatingthe interaction of the protein with other biological molecules, as inthe case of receptor binding sites.

A second and equally important complication of existing methods forprotein PEGylation is that the PEG derivatives can undergo undesiredside reactions with residues other than those desired. Histidinecontains a reactive imino moiety, represented structurally as —N(H)—,but many chemically reactive species that react with epsilon —NH₂ canalso react with —N(H)—. Similarly, the side chain of the amino acidcysteine bears a free sulfhydryl group, represented structurally as —SH.In some instances, the PEG derivatives directed at the epsilon —NH₂group of lysine also react with cysteine, histidine or other residues.This can create complex, heterogeneous mixtures of PEG-derivatizedbioactive molecules and risks destroying the activity of the bioactivemolecule being targeted. It would be desirable to develop PEGderivatives that permit a chemical functional group to be introduced ata single site within the protein that would then enable the selectivecoupling of one or more PEG polymers to the bioactive molecule atspecific sites on the protein surface that are both well-defined andpredictable.

In addition to lysine residues, considerable effort in the art has beendirected toward the development of activated PEG reagents that targetother amino acid side chains, including cysteine, histidine and theN-terminus. See, e.g., U.S. Pat. No. 6,610,281 which is incorporated byreference herein, and “Polyethylene Glycol and Derivatives for AdvancedPEGylation”, Nektar Molecular Engineering Catalog, 2003, pp. 1-17. Acysteine residue can be introduced site-selectively into the structureof proteins using site-directed mutagenesis and other techniques knownin the art, and the resulting free sulfhydryl moiety can be reacted withPEG derivatives that bear thiol-reactive functional groups. Thisapproach is complicated, however, in that the introduction of a freesulfhydryl group can complicate the expression, folding and stability ofthe resulting protein. Thus, it would be desirable to have a means tointroduce a chemical functional group into bioactive molecules thatenables the selective coupling of one or more PEG polymers to theprotein while simultaneously being compatible with (i.e., not engagingin undesired side reactions with) sulfhydryls and other chemicalfunctional groups typically found in proteins.

As can be seen from a sampling of the art, many of these derivativesthat have been developed for attachment to the side chains of proteins,in particular, the —NH₂ moiety on the lysine amino acid side chain andthe —SH moiety on the cysteine side chain, have proven problematic intheir synthesis and use. Some form unstable linkages with the proteinthat are subject to hydrolysis and therefore decompose, degrade, or areotherwise unstable in aqueous environments, such as in the bloodstream.See Pedder, S. C. Semin Liver Dis. 2003; 23 Suppl 1:19-22 for adiscussion of the stability of linkages in PEG-Intron®. Some form morestable linkages, but are subject to hydrolysis before the linkage isformed, which means that the reactive group on the PEG derivative may beinactivated before the protein can be attached. Some are somewhat toxicand are therefore less suitable for use in vivo. Some are too slow toreact to be practically useful. Some result in a loss of proteinactivity by attaching to sites responsible for the protein's activity.Some are not specific in the sites to which they will attach, which canalso result in a loss of desirable activity and in a lack ofreproducibility of results. In order to overcome the challengesassociated with modifying proteins with poly(ethylene glycol) moieties,PEG derivatives have been developed that are more stable (e.g., U.S.Pat. No. 6,602,498, which is incorporated by reference herein) or thatreact selectively with thiol moieties on molecules and surfaces (e.g.,U.S. Pat. No. 6,610,281, which is incorporated by reference herein).There is clearly a need in the art for PEG derivatives that arechemically inert in physiological environments until called upon toreact selectively to form stable chemical bonds.

Recently, an entirely new technology in the protein sciences has beenreported, which promises to overcome many of the limitations associatedwith site-specific modifications of proteins. Specifically, newcomponents have been added to the protein biosynthetic machinery of theprokaryote Escherichia coli (E. coli) (e.g., L. Wang, et al., (2001),Science 292:498-500) and the eukaryote Saccharomyces cerevisiae (S.cerevisiae) (e.g., J. Chin et al., Science 301:964-7 (2003)), which hasenabled the incorporation of non-genetically encoded amino acids toproteins in vivo. A number of new amino acids with novel chemical,physical or biological properties, including photoaffinity labels andphotoisomerizable amino acids, photocrosslinking amino acids (see, e.g.,Chin, J. W., et al. (2002) Proc. Natl. Acad. Sci. U.S. A.99:11020-11024; and, Chin, J. W., et al., (2002) J. Am. Chem. Soc.124:9026-9027), keto amino acids, heavy atom containing amino acids, andglycosylated amino acids have been incorporated efficiently and withhigh fidelity into proteins in E. coli and in yeast in response to theamber codon, TAG, using this methodology. See, e.g., J. W. Chin et al.,(2002), Journal of the American Chemical Society 124:9026-9027; J. W.Chin, & P. G. Schultz, (2002), Chem Bio Chem 3(11):1135-1137; J. W.Chin, et al., (2002), PNAS United States of America 99:11020-11024; and,L. Wang, & P. G. Schultz, (2002), Chem. Comm., 1:1-11. All referencesare incorporated by reference in their entirety. These studies havedemonstrated that it is possible to selectively and routinely introducechemical functional groups, such as ketone groups, alkyne groups andazide moieties, that are not found in proteins, that are chemicallyinert to all of the functional groups found in the 20 common,genetically-encoded amino acids and that may be used to reactefficiently and selectively to form stable covalent linkages.

The ability to incorporate non-genetically encoded amino acids intoproteins permits the introduction of chemical functional groups thatcould provide valuable alternatives to the naturally-occurringfunctional groups, such as the epsilon —NH₂ of lysine, the sulfhydryl—SH of cysteine, the imino group of histidine, etc. Certain chemicalfunctional groups are known to be inert to the functional groups foundin the 20 common, genetically-encoded amino acids but react cleanly andefficiently to form stable linkages. Azide and acetylene groups, forexample, are known in the art to undergo a Huisgen [3+2]cycloadditionreaction in aqueous conditions in the presence of a catalytic amount ofcopper. See, e.g., Tomoe, et al., (2002) J. Org. Chem. 67:3057-3064;and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599. Byintroducing an azide moiety into a protein structure, for example, oneis able to incorporate a functional group that is chemically inert toamines, sulfhydryls, carboxylic acids, hydroxyl groups found inproteins, but that also reacts smoothly and efficiently with anacetylene moiety to form a cycloaddition product. Importantly, in theabsence of the acetylene moiety, the azide remains chemically inert andunreactive in the presence of other protein side chains and underphysiological conditions.

The present invention addresses, among other things, problems associatedwith the activity and production of interferon polypeptides, and alsoaddresses the production of an interferon polypeptide with improvedbiological or pharmacological properties, such as improved therapeutichalf-life and/or modulation of one or more biological activities or sideeffects found with current IFN therapeutics.

BRIEF SUMMARY OF THE INVENTION

This invention provides hIFN polypeptides comprising one or morenon-naturally encoded amino acids.

In some embodiments, the hIFN polypeptide comprises one or morepost-translational modifications. In some embodiments, the hIFNpolypeptide is linked to a linker, polymer, or biologically activemolecule. In some embodiments, the hIFN polypeptide is linked to abifunctional polymer, bifunctional linker, or at least one additionalhIFN polypeptide.

In some embodiments, the non-naturally encoded amino acid is linked to awater soluble polymer. In some embodiments, the water soluble polymercomprises a poly(ethylene glycol) moiety. In some embodiments, thenon-naturally encoded amino acid is linked to the water soluble polymerwith a linker or is bonded to the water soluble polymer. In someembodiments, the poly(ethylene glycol) molecule is a bifunctionalpolymer. In some embodiments, the bifunctional polymer is linked to asecond polypeptide. In some embodiments, the second polypeptide is ahIFN polypeptide. In some embodiments, the hIFN polypeptide comprisesone or more naturally-encoded amino acid substitutions, additions, ordeletions. In some embodiments, the hIFN polypeptide comprises one ormore naturally encoded amino acid substitutions.

In some embodiments, the hIFN polypeptide comprises at least two aminoacids linked to a water soluble polymer comprising a poly(ethyleneglycol) moiety. In some embodiments, at least one amino acid is anon-naturally encoded amino acid.

In some embodiments, one or more non-naturally encoded amino acids areincorporated at any position in one or more of the following regionscorresponding to secondary structures in IFN as follows: 1-9(N-terminus), 10-21 (A helix), 22-39 (region between A helix and Bhelix), 40-75 (B helix), 76-77 (region between B helix and C helix),78-100 (C helix), 101-110 (region between C helix and D helix), 111-132(D helix), 133-136 (region between D and E helix), 137-155 (E helix),156-165 (C-terminus) (SEQ ID NO: 2, or the corresponding amino acids inSEQ ID NO: 1 or 3, other interferons, or interferon-like cytokines suchas limitin). In some embodiments, one or more non-naturally encodedamino acid are substituted at, but not limited to, one or more of thefollowing positions of hIFN (as in SEQ ID NO: 2, or the correspondingamino acid in SEQ ID NO: 1, 3, or any other IFN polypeptide): beforeposition 1 (i.e., at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, or 166 (i.e. at thecarboxyl terminus). In some embodiments, one or more non-naturallyencoded amino acids are incorporated in one or more of the followingpositions in IFN: before position 1 (i.e. at the N terminus), 1, 2, 3,4, 5, 6, 7, 8, 9, 12, 13, 16, 19, 20, 22, 23, 24, 25, 26, 27, 28, 30,31, 32, 33, 34, 35, 40, 41, 42, 45, 46, 48, 49, 50, 51, 58, 61, 64, 65,68, 69, 70, 71, 73, 74, 77, 78, 79, 80, 81, 82, 83, 85, 86, 89, 90, 93,94, 96, 97, 100, 101, 103, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 117, 118, 120, 121, 124, 125, 127, 128, 129, 131, 132, 133, 134,135, 136, 137, 148, 149, 152, 153, 156, 158, 159, 160, 161, 162, 163,164, 165, 166 (i.e. at the carboxyl terminus of the protein) (SEQ ID NO:2, or the corresponding amino acids in SEQ ID NO: 1 or 3). In someembodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in IFN: 6, 9, 12,13, 16, 41, 45, 46, 48, 49, 61, 64, 65, 96, 100, 101, 103, 106, 107,108, 110, 111, 113, 114, 117, 120, 121, 149, 156, 159, 160, 161 and 162(SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 1 or 3).In some embodiments, the IFN polypeptides of the invention comprise oneor more non-naturally encoded amino acids at one or more of thefollowing positions: 100, 106, 107, 108, 111, 113, 114 (SEQ ID NO: 2, orthe corresponding amino acids in SEQ ID NO: 1 or 3). In someembodiments, the IFN polypeptides of the invention comprise one or morenon-naturally encoded amino acids at one or more of the followingpositions: 41, 45, 46, 48, 49 (SEQ ID NO: 2, or the corresponding aminoacids in SEQ ID NO: 1 or 3). In some embodiments, the IFN polypeptidesof the invention comprise one or more non-naturally encoded amino acidsat one or more of the following positions: 61, 64, 65, 101, 103, 110,117, 120, 121, 149 (SEQ ID NO: 2, or the corresponding amino acids inSEQ ID NO: 1 or 3). In some embodiments, the IFN polypeptides of theinvention comprise one or more non-naturally encoded amino acids at oneor more of the following positions: 6, 9, 12, 13, 16, 96, 156, 159, 160,161, 162 (SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 1or 3). In some embodiments, the IFN polypeptides of the inventioncomprise one or more non-naturally encoded amino acids at one or more ofthe following positions: 2, 3, 4, 5, 7, 8, 16, 19, 20, 40, 42, 50, 51,58, 68, 69, 70, 71, 73, 97, 105, 109, 112, 118, 148, 149, 152, 153, 158,163, 164, 165 (SEQ ID NO: 2, or the corresponding amino acids in SEQ IDNO: 1 or 3). In some embodiments, the IFN polypeptides of the inventioncomprise one or more non-naturally encoded amino acids at one or more ofthe following positions: 34, 78, 107 (SEQ ID NO: 2, or the correspondingamino acid in SEQ ID NO: 1, 3, or any other IFN polypeptide). In someembodiments, the non-naturally encoded amino acid at one or more ofthese or other positions is linked to a water soluble polymer, includingbut not limited to positions: before position 1 (i.e., at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, or 166 (i.e. at the carboxyl terminus) (SEQ ID NO:2, or the corresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide). In some embodiments, the non-naturally encoded amino acidat one or more of these positions is linked to a water soluble polymer,including but not limited to positions: before position 1 (i.e. at the Nterminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 13, 16, 19, 20, 22, 23, 24,25, 26, 27, 28, 30, 31, 32, 33, 34, 35, 40, 41, 42, 45, 46, 48, 49, 50,51, 58, 61, 64, 65, 68, 69, 70, 71, 73, 74, 77, 78, 79, 80, 81, 82, 83,85, 86, 89, 90, 93, 94, 96, 97, 100, 101, 103, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 117, 118, 120, 121, 124, 125, 127, 128, 129,131, 132, 133, 134, 135, 136, 137, 148, 149, 152, 153, 156, 158, 159,160, 161, 162, 163, 164, 165, 166 (i.e. at the carboxyl terminus) (SEQID NO: 2, or the corresponding amino acids in SEQ ID NO: 1 or 3). Insome embodiments, the non-naturally encoded amino acid is linked to awater soluble polymer at one or more of the following positions: 6, 9,12, 13, 16, 41, 45, 46, 48, 49, 61, 64, 65, 96, 100, 101, 103, 106, 107,108, 110, 111, 113, 114, 117, 120, 121, 149, 156, 159, 160, 161 and 162(SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 1 or 3).In some embodiments, the non-naturally encoded amino acid is linked to awater soluble polymer at one or more of the following positions: 100,106, 107, 108, 111, 113, 114 (SEQ ID NO: 2, or the corresponding aminoacids in SEQ ID NO: 1 or 3). In some embodiments, the non-naturallyencoded amino acid is linked to a water soluble polymer at one or moreof the following positions: 41, 45, 46, 48, 49 (SEQ ID NO: 2, or thecorresponding amino acids in SEQ ID NO: 1 or 3). In some embodiments,the non-naturally encoded amino acid is linked to a water solublepolymer at one or more of the following positions: 61, 64, 65, 101, 103,110, 117, 120, 121, 149 (SEQ ID NO: 2, or the corresponding amino acidsin SEQ ID NO: 1 or 3). In some embodiments, the non-naturally encodedamino acid is linked to a water soluble polymer at one or more of thefollowing positions: 6, 9, 12, 13, 16, 96, 156, 159, 160, 161, 162 (SEQID NO: 2, or the corresponding amino acids in SEQ ID NO: 1 or 3).

In some embodiments, the one or more non-naturally encoded amino acidsat one or more of the following positions is linked to one or morewater-soluble polymer: 2, 3, 4, 5, 7, 8, 16, 19, 20, 40, 42, 50, 51, 58,68, 69, 70, 71, 73, 97, 105, 109, 112, 118, 148, 149, 152, 153, 158,163, 164, 165 (SEQ ID NO: 2, or the corresponding amino acid in or thecorresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide). In some embodiments, the one or more non-naturally encodedamino acids at one or more of the following positions is linked to oneor more water-soluble polymer: 34, 78, 107 (SEQ ID NO: 2, or thecorresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide). In some embodiments, the water soluble polymer is coupledto the IFN polypeptide to a non-naturally encoded amino acid at one ormore of the following amino acid positions: 6, 9, 12, 13, 16, 41, 45,46, 48, 49, 61, 64, 65, 96, 100, 101, 103, 106, 107, 108, 110, 111, 113,114, 117, 120, 121, 149, 156, 159, 160, 161 and 162 (SEQ ID NO: 2, orthe corresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide). In some embodiments the water soluble polymer is coupledto the IFN polypeptide at one or more of the following amino acidpositions: 6, 9, 12, 13, 16, 41, 45, 46, 48, 49, 61, 64, 65, 96, 100,101, 103, 106, 107, 108, 110, 111, 113, 114, 117, 120, 121, 149, 156,159, 160, 161 and 162 (SEQ ID NO: 2, or the corresponding amino acid inSEQ ID NO: 1, 3, or any other IFN polypeptide). In some embodiments, thenon-naturally encoded amino acid at one or more of these positions islinked to one or more water soluble polymers, positions: 34, 78, 107(SEQ ID NO: 2, or the corresponding amino acid in or the correspondingamino acid in SEQ ID NO: 1, 3, or any other IFN polypeptide). In someembodiments, the IFN polypeptides of the invention comprise one or morenon-naturally encoded amino acids at one or more of the followingpositions providing an antagonist: 2, 3, 4, 5, 7, 8, 16, 19, 20, 40, 42,50, 51, 58, 68, 69, 70, 71, 73, 97, 105, 109, 112, 118, 148, 149, 152,153, 158, 163, 164, 165 (SEQ ID NO: 2, or the corresponding amino acidin or the corresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide); a hIFN polypeptide comprising one of these substitutionsmay potentially act as a weak antagonist or weak agonist depending onthe intended site selected and desired activity. Human IFN antagonistsinclude, but are not limited to, hIFN polypeptides with one or morenon-naturally encoded amino acid substitutions at 22, 23, 24, 25, 26,27, 28, 30, 31, 32, 33, 34, 35, 74, 77, 78, 79, 80, 82, 83, 85, 86, 89,90, 93, 94, 124, 125, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137,or any combinations thereof (hIFN; SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NO: 1 or 3, or any other IFN polypeptide).

In some embodiments, one or more non-naturally encoded amino acids aresubstituted at, but not limited to, one or more of the followingpositions of hIFN (as in SEQ ID NO: 2, or the corresponding amino acidsin SEQ ID NO: 1 or 3, or any other IFN polypeptide): 31, 134, 34, 38,129, 36, 122, 37, 121, 41, 125, 124, 149, 117, 39, 118, 120, 107, 108,106, 100, 111, 113, 114, 41, 45, 46, 48, 49, 61, 64, 65, 101, 103, 102,110, 117, 120, 121, 149, 96, 6, 9, 12, 13, 16, 68, 70, 109, 159, 161,156, 160, 162, 24, 27, 78, 83, 85, 87, 89, 164. In one embodiment, anon-naturally encoded amino acid is substituted at position 38 of hIFN(as in SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 1 or3, or any other IFN polypeptide). In some embodiments, the non-naturallyencoded amino acid at one or more of these or other positions is linkedto a water soluble polymer, including but not limited to positions: 31,134, 34, 38, 129, 36, 122, 37, 121, 41, 125, 124, 149, 117, 39, 118,120, 107, 108, 106, 100, 111, 113, 114, 41, 45, 46, 48, 49, 61, 64, 65,101, 103, 102, 110, 117, 120, 121, 149, 96, 6, 9, 12, 13, 16, 68, 70,109, 159, 161, 156, 160, 162, 24, 27, 78, 83, 85, 87, 89, 164 (as in SEQID NO: 2, or the corresponding amino acids in SEQ ID NO: 1 or 3, or anyother IFN polypeptide).

In some embodiments, one or more non-naturally encoded amino acids aresubstituted at, but not limited to, one or more of the followingpositions of hIFN (SEQ ID NO: 2 or the corresponding amino acids in SEQID NO: 1 or 3): before position 1 (i.e., at the N-terminus), 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, or 166(i.e. at the carboxyl terminus) and one or more natural amino acidsubstitutions. In some embodiments, the one or more non-naturallyencoded amino acids are coupled to a water soluble polymer. In someembodiments, the one or more non-naturally encoded amino acids arecoupled to PEG. In one embodiment, the natural amino acid substitutionis R149Y. In some embodiments, the natural amino acid substitution isR149E. In some embodiments, the natural amino acid substitution isR149S. In one embodiment, the non-natural amino acid substitution is atposition 107 and the natural amino acid substitution is R149Y. In oneembodiment, the non-natural amino acid substitution is at position 106and the natural amino acid substitution is R149Y. In some embodiments,the one or more naturally encoded amino acid substitution is at one ormore of the following positions of hIFN (SEQ ID NO: 2 or thecorresponding amino acids in SEQ ID NO; 1 or 3), including but notlimited to: 10, 16, 13, 79, 83, 85, 86, 87, 90, 91, 93, 94, 96, 120,121, 124, 125, 128, 149. In some embodiments, the one or more naturallyencoded amino acid substitution is one or more of the followingsubstitutions (SEQ ID NO: 2 or the corresponding amino acids in SEQ IDNO: 1 or 3), including but not limited to: G10E, M16R, R13E, T79R, K83Q,K83S, Y85L, T86S, E87S, Q90R, Q91E, N93Q, D94V, E96K, R120K, K121T,Q124R, R125G, L128R, R149Y, R149E, R149S. In some embodiments, thenatural amino acid substitution is at position 1 (the N-terminus). Insome embodiments, one or more non-naturally encoded amino acids aresubstituted at one or more of the following positions of hIFN (as in SEQID NO: 2, or the corresponding amino acids in other IFN's): 107, 78, 34.In some embodiments, the non-naturally encoded amino acid at one or moreof these positions is coupled to a water soluble polymer: 107, 78, 34.

One or more amino acids found in a limitin sequence may be substitutedinto a hIFN polypeptide (hybrid limitin/hIFN polypeptides). Examplesinclude but are not limited to the natural amino acid substitutionsdescribed in the previous paragraph. Alternatively, a set of amino acidsfound in an interferon polypeptide may be replaced by a set of aminoacids found in a limitin sequence. A set of amino acids may comprisecontiguous amino acids or amino acids present in different portions ofthe molecule but are involved in a structural characteristic orbiological activity of the polypeptide. The mouse limitin molecule hasan improved CFU-GM toxicity profile compared to other IFNα proteins.Alignment of human IFNα-2a with the limitin protein sequence showed 30%amino acid identity. 50% sequence conservation was also observed. Inparticular, a prominent deletion in the limitin sequence between the Cand D helices (in the loop between C and D helices) was observed. The“HV” mutant was generated with the following substitutions in hIFNα-2a(SEQ ID NO: 2): D77-D94 is replaced with the mouse limitin sequenceHERALDQLLSSLWRELQV. The “CD” mutant was generated with the followingsubstitutions in hIFNα-2a (SEQ ID NO: 2): V105-D114 with GQSAPLP. Thishybrid molecule with the loop region from limitin substituted into thehuman IFNα-2a protein (“CD” mutant) was found to have equivalentanti-viral activity as the WHO IFN standard. In addition to the one ormore limitin amino acids, the hIFN polypeptide may comprise one or morenon-naturally encoded amino acids at any one or more positions of thehIFN polypeptide. In some embodiments, the one or more non-naturallyencoded amino acids may be linked to a water soluble polymer such as PEGor bonded directly to a water soluble polymer such as PEG. In additionto the natural amino acid substitutions in the HV or the CD mutant, oneor more additional natural amino acid substitutions may be found in thehIFN polypeptide.

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 6, 16, 34, 39, 45, 46, 64, 65, 68, 78, 85, 87, 101, 107, 108,111, 114, 118, 124, 125, 145, 146, 153, 156, 96, 149 (SEQ ID NO: 2, orthe corresponding amino acid in or the corresponding amino acid in SEQID NO: 1, 3, or any other IFN polypeptide). In some embodiments, thehIFN polypeptide comprises one or more non-naturally encoded amino acidsat one or more of the following positions linked to one or morewater-soluble polymer: 6, 16, 34, 39, 45, 46, 64, 65, 68, 78, 85, 87,101, 107, 108, 111, 114, 118, 124, 125, 145, 146, 153, 156, 96, 149 (SEQID NO: 2, or the corresponding amino acid in or the corresponding aminoacid in SEQ ID NO: 1, 3, or any other IFN polypeptide). In someembodiments, the hIFN polypeptide comprises one or more non-naturallyencoded amino acids at one or more of the following positions linked toone or more water-soluble polymer: 6, 16, 34, 39, 45, 46, 64, 65, 68,78, 85, 87, 101, 107, 108, 111, 114, 118, 124, 125, 145, 146, 153, 156,96, 149 (SEQ ID NO: 2, or the corresponding amino acid in or thecorresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide) and comprises one or more naturally encoded amino acidsubstitution. In some embodiments, the hIFN polypeptide comprises one ormore non-naturally encoded amino acids at one or more of the followingpositions linked to one or more water-soluble polymer: 6, 16, 34, 39,45, 46, 64, 65, 68, 78, 85, 87, 101, 107, 108, 111, 114, 118, 124, 125,145, 146, 153, 156, 96, 149 (SEQ ID NO: 2, or the corresponding aminoacid in or the corresponding amino acid in SEQ ID NO: 1, 3, or any otherIFN polypeptide) and comprises one or more of the following naturallyencoded amino acid substitutions G10E, M16R, R13E, T79R, K83Q, K83S,Y85L, T86S, E87S, Q90R, Q91E, N93Q, D94V, E96K, R120K, K121T, Q124R,R125G, L128R, R149Y, R149E, R149S.

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 6, 16, 37, 45, 46, 78, 87, 89, 107, 108 (SEQ ID NO: 2 or thecorresponding amino acids in SEQ ID NO: 1 or 3). In some embodiments,the hIFN polypeptide comprises one or more non-naturally encoded aminoacids at one or more of the following positions: 6, 16, 37, 45, 46, 78,87, 89, 107, 108 (SEQ ID NO: 2 or the corresponding amino acids in SEQID NO: 1 or 3) that is linked to a water soluble polymer. In someembodiments, the hIFN polypeptide comprises one or more non-naturallyencoded amino acids at one or more of the following positions: 6, 16,37, 45, 46, 78, 87, 89, 107, 108 (SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NO: 1 or 3) that is bonded to a water solublepolymer.

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 6, 16, 37, 45, 46, 78, 87, 89, 107, 108 and one or more ofthe following naturally encoded amino acid substitutions: T79R, L80A,K83S, Y85L, Y85S, T86S, E87S, Q91E (SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NO: 1 or 3). In some embodiments, the hIFNpolypeptide comprises one or more non-naturally encoded amino acids atone or more of the following positions: 6, 16, 37, 45, 46, 78, 87, 89,107, 108 that is linked to a water soluble polymer and comprises one ormore of the following naturally encoded amino acid substitutions: T79R,L80A, K83S, Y85L, Y85S, T86S, E87S, Q91E (SEQ ID NO: 2 or thecorresponding amino acids in SEQ ID NO: 1 or 3). In some embodiments,the hIFN polypeptide comprises one or more non-naturally encoded aminoacids at one or more of the following positions: 6, 16, 37, 45, 46, 78,87, 89, 107, 108 that is bonded to a water soluble polymer and comprisesone or more of the following naturally encoded amino acid substitutions:T79R, L80A, K83S, Y85L, Y85S, T86S, E87S, Q91E (SEQ ID NO: 2 or thecorresponding amino acids in SEQ ID NO: 1 or 3).

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 6, 16, 37, 45, 46, 78, 87, 107, and 108 (SEQ ID NO: 2 or thecorresponding amino acids in SEQ ID NO: 1 or 3). In some embodiments,the hIFN polypeptide comprises one or more non-naturally encoded aminoacids at one or more of the following positions: 6, 16, 37, 45, 46, 78,87, 107, and 108 (SEQ ID NO: 2 or the corresponding amino acids in SEQID NO: 1 or 3) that is linked to a water soluble polymer. In someembodiments, the hIFN polypeptide comprises one or more non-naturallyencoded amino acids at one or more of the following positions: 6, 16,37, 45, 46, 78, 87, 107, and 108 (SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NO: 1 or 3) that is bonded to a water solublepolymer.

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 6, 16, 37, 45, 46, 78, 87, 107, and 108 and comprises one ormore of the following naturally encoded amino acid substitutions: T79R,K83S, Y85L, T86S, E87S, Q91E (SEQ ID NO: 2 or the corresponding aminoacids in SEQ ID NO: 1 or 3). In some embodiments, the hIFN polypeptidecomprises one or more non-naturally encoded amino acids at one or moreof the following positions: 6, 16, 37, 45, 46, 78, 87, 107, and 108 thatis linked to a water soluble polymer and comprises one or more of thefollowing naturally encoded amino acid substitutions: T79R, K83S, Y85L,T86S, E87S, Q91E (SEQ ID NO: 2 or the corresponding amino acids in SEQID NO: 1 or 3). In some embodiments, the hIFN polypeptide comprises oneor more non-naturally encoded amino acids at one or more of thefollowing positions: 6, 16, 37, 45, 46, 78, 87, 107, and 108 that isbonded to a water soluble polymer and comprises one or more of thefollowing naturally encoded amino acid substitutions: T79R, K83S, Y85L,T86S, E87S, Q91E (SEQ ID NO: 2 or the corresponding amino acids in SEQID NO: 1 or 3).

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 37, 45, 46, 89, and 107 (SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NO: 1 or 3). In some embodiments, the hIFNpolypeptide comprises one or more non-naturally encoded amino acids atone or more of the following positions: 37, 45, 46, 89, and 107 (SEQ IDNO: 2 or the corresponding amino acids in SEQ ID NO: 1 or 3) that islinked to a water soluble polymer. In some embodiments, the hIFNpolypeptide comprises one or more non-naturally encoded amino acids atone or more of the following positions: 37, 45, 46, 89, and 107 (SEQ IDNO: 2 or the corresponding amino acids in SEQ ID NO: 1 or 3) that isbonded to a water soluble polymer.

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 37, 45, 46, 89, and 107 and comprises one or more of thefollowing naturally encoded amino acid substitutions: T79R, L80A, Y85L,Y85S, E87S (SEQ ID NO: 2 or the corresponding amino acids in SEQ ID NO:1 or 3). In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 37, 45, 46, 89, and 107 that is linked to a water solublepolymer and comprises one or more of the following naturally encodedamino acid substitutions: T79R, L80A, Y85L, Y85S, E87S (SEQ ID NO: 2 orthe corresponding amino acids in SEQ ID NO: 1 or 3). In someembodiments, the hIFN polypeptide comprises one or more non-naturallyencoded amino acids at one or more of the following positions: 37, 45,46, 89, and 107 that is bonded to a water soluble polymer and comprisesone or more of the following naturally encoded amino acid substitutions:T79R, L80A, Y85L, Y85S, E87S (SEQ ID NO: 2 or the corresponding aminoacids in SEQ ID NO: 1 or 3).

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 23, 24, 25, 26, 27, 28, 30, 31, 32, 33, 128, 129, 131, 132,133, 134, 135, 136, 137, 158, 159, 160, 161, 162, 163, 164, 165 (SEQ IDNO: 2 or the corresponding amino acids in SEQ ID NO: 1 or 3). In someembodiments, the hIFN polypeptide comprises one or more non-naturallyencoded amino acids at one or more of the following positions that islinked or bonded to a water soluble polymer: 23, 24, 25, 26, 27, 28, 30,31, 32, 33, 128, 129, 131, 132, 133, 134, 135, 136, 137, 158, 159, 160,161, 162, 163, 164, 165 (SEQ ID NO: 2 or the corresponding amino acidsin SEQ ID NO: 1 or 3). In some embodiments, the hIFN polypeptidecomprises one or more non-naturally encoded amino acids at one or moreof the following positions: 23, 24, 27, 31, 128, 131, 134, 158 (SEQ IDNO: 2 or the corresponding amino acids in SEQ ID NO: 1 or 3). In someembodiments, the hIFN polypeptide comprises one or more non-naturallyencoded amino acids at one or more of the following positions that islinked or bonded to a water soluble polymer: 23, 24, 27, 31, 128, 131,134, 158 (SEQ ID NO: 2 or the corresponding amino acids in SEQ ID NO: 1or 3). In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 24, 27, 31, 128, 131, 134 (SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NO: 1 or 3). In some embodiments, the hIFNpolypeptide comprises one or more non-naturally encoded amino acids atone or more of the following positions that is linked or bonded to awater soluble polymer: 24, 27, 31, 128, 131, 134 (SEQ ID NO: 2 or thecorresponding amino acids in SEQ ID NO: 1 or 3).

In some embodiments, the IFN polypeptides of the invention comprise oneor more non-naturally encoded amino acids at one or more of thefollowing positions providing an antagonist: 2, 3, 4, 5, 7, 8, 16, 19,20, 40, 42, 50, 51, 58, 68, 69, 70, 71, 73, 97, 105, 109, 112, 118, 148,149, 152, 153, 158, 163, 164, 165, or any combination thereof (SEQ IDNO: 2, or the corresponding amino acids in SEQ ID NO: 1 or 3); a hIFNpolypeptide comprising one of these substitutions may potentially act asa weak antagonist or weak agonist depending on the site selected anddesired activity. Human IFN antagonists include, but are not limited to,those with one or more substitutions at 22, 23, 24, 25, 26, 27, 28, 30,31, 32, 33, 34, 35, 74, 77, 78, 79, 80, 82, 83, 85, 86, 89, 90, 93, 94,124, 125, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137, or anycombination thereof (hIFN; SEQ ID NO: 2 or the corresponding amino acidsin SEQ ID NO: 1 or 3).

In some embodiments, one or more non-naturally encoded amino acids aresubstituted at, but not limited to, one or more of the followingpositions of hIFN (as in SEQ ID NO: 2, or the corresponding amino acidsin other IFN's): 31, 134, 34, 38, 129, 36, 122, 37, 121, 41, 125, 124,149, 117, 39, 118, 120, 107, 108, 106, 100, 111, 113, 114, 41, 45, 46,48, 49, 61, 64, 65, 101, 103, 102, 110, 117, 120, 121, 149, 96, 6, 9,12, 13, 16, 68, 70, 109, 159, 161, 156, 160, 162, 24, 27, 78, 83, 85,87, 89, 164. In one embodiment, a non-naturally encoded amino acid issubstituted at position 38 of hIFN (as in SEQ ID NO: 2, or thecorresponding amino acids in other IFN's). In some embodiments, thenon-naturally encoded amino acid at these or other positions is linkedto a water soluble polymer, including but not limited to positions: 31,134, 34, 38, 129, 36, 122, 37, 121, 41, 125, 124, 149, 117, 39, 118,120, 107, 108, 106, 100, 111, 113, 114, 41, 45, 46, 48, 49, 61, 64, 65,101, 103, 102, 110, 117, 120, 121, 149, 96, 6, 9, 12, 13, 16, 68, 70,109, 159, 161, 156, 160, 162, 24, 27, 78, 83, 85, 87, 89, 164.

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acid and a naturally encoded amino acidsubstitution. In some embodiments, the non-naturally encoded amino acidpresent in the hIFN polypeptide is linked to a water soluble polymer andthe hIFN polypeptide comprises one or more naturally encoded amino acidsubstitution.

In some embodiments, the hIFN polypeptide comprises a substitution,addition or deletion that modulates affinity of the hIFN polypeptide fora hIFN polypeptide receptor. In some embodiments, the hIFN polypeptidecomprises a substitution, addition, or deletion that increases thestability of the hIFN polypeptide when compared with the stability ofthe corresponding hIFN without the substitution, addition, or deletion.In some embodiments, the hIFN polypeptide comprises a substitution,addition, or deletion that modulates the immunogenicity of the hIFNpolypeptide when compared with the immunogenicity of the correspondinghIFN without the substitution, addition, or deletion. In someembodiments, the hIFN polypeptide comprises a substitution, addition, ordeletion that modulates serum half-life or circulation time of the hIFNpolypeptide when compared with the serum half-life or circulation timeof the corresponding hIFN without the substitution, addition, ordeletion.

In some embodiments, the hIFN polypeptide comprises a substitution,addition, or deletion that modulates hIFN polypeptide receptorconformation when compared with hIFN polypeptide receptor conformationwith the corresponding hIFN without the substitution, addition, ordeletion. In some embodiments, the hIFN polypeptide comprises asubstitution, addition, or deletion that modulates one or moredownstream signaling events of the hIFN polypeptide receptor whencompared to the downstream signaling events of the hIFN polypeptidereceptor with the corresponding hIFN without the substitution, addition,or deletion. In some embodiments, the hIFN polypeptide comprises asubstitution, addition, or deletion that modulates hIFN polypeptidereceptor binding kinetics when compared to hIFN polypeptide receptorbinding kinetics with the corresponding hIFN without the substitution,addition, or deletion.

In some embodiments, the hIFN polypeptide comprises a substitution,addition, or deletion that increases the aqueous solubility of the hIFNpolypeptide when compared to aqueous solubility of the correspondinghIFN without the substitution, addition, or deletion. In someembodiments, the hIFN polypeptide comprises a substitution, addition, ordeletion that increases the solubility of the hIFN polypeptide producedin a host cell when compared to the solubility of the corresponding hIFNwithout the substitution, addition, or deletion. In some embodiments,the hIFN polypeptide comprises a substitution, addition, or deletionthat increases the expression of the hIFN polypeptide in a host cell orincreases synthesis in vitro when compared to the expression orsynthesis of the corresponding hIFN without the substitution, addition,or deletion. In some embodiments, the hIFN polypeptide comprises asubstitution, addition, or deletion that increases protease resistanceof the hIFN polypeptide when compared to the protease resistance of thecorresponding hIFN without the substitution, addition, or deletion. Insome embodiments, the hIFN polypeptide comprises a substitution,addition, or deletion that modulates interaction with one or moremembers of the opioid family of receptors. In some embodiments, the hIFNpolypeptide comprises a substitution, addition, or deletion thatmodulates 5-HT brain neurotransmission. In some embodiments, the hIFNpolypeptide comprises a substitution, addition, or deletion thatmodulates induction of indoleamine 2,3-dioxygenase (IDO). In someembodiments, the hIFN polypeptide comprises a substitution, addition, ordeletion that modulates one or more of the biological activities of IFN,including but not limited to, side effects found with current IFNtherapeutics. In some embodiments, the hIFN polypeptide comprises asubstitution, addition, or deletion that has modulated toxicity whencompared to the toxicity of the corresponding hIFN without thesubstitution, addition, or deletion. In some embodiments, the hIFNpolypeptide comprises a non-naturally encoded amino acid linked to awater soluble polymer that modulates one or more side effects found withIFN. In some embodiments, the hIFN polypeptide comprises a non-naturallyencoded amino acid linked to a water soluble polymer and has modulatedtoxicity. In some embodiments, the hIFN polypeptide comprises asubstitution, addition, deletion, or non-naturally encoded amino acidthat has modulated anti-viral activity when compared to the anti-viralactivity of the corresponding hIFN without the substitution, addition,deletion, or non-naturally encoded amino acid. In some embodiments, thehIFN polypeptide comprises a substitution, addition, deletion, ornon-naturally encoded amino acid that has modulated immunogenicity whencompared to the immunogenicity of the corresponding hIFN without thesubstitution, addition, deletion, or non-naturally encoded amino acid.In some embodiments, the hIFN polypeptide comprises a substitution,addition, deletion, or non-naturally encoded amino acid that hasmodulated anti-tumor activity when compared to the anti-tumor activityof the corresponding hIFN without the substitution, addition, deletion,or non-naturally encoded amino acid. In some embodiments, the hIFNpolypeptide comprises a substitution, addition, deletion, ornon-naturally encoded amino acid that has modulated anti-infectiveactivity when compared to the anti-infective activity of thecorresponding hIFN without the substitution, addition, deletion, ornon-naturally encoded amino acid. In some embodiments, the hIFNpolypeptide comprises a substitution, addition, deletion, ornon-naturally encoded amino acid that has modulated prophylaxis activityfor infectious agents when compared to the prophylaxis activity forinfectious agents of the corresponding hIFN without the substitution,addition, deletion, or non-naturally encoded amino acid. In someembodiments, the hIFN polypeptide comprises a substitution, addition,deletion, or non-naturally encoded amino acid that has modulated tumorprophylaxis activity when compared to the tumor prophylaxis activity ofthe corresponding hIFN without the substitution, addition, deletion, ornon-naturally encoded amino acid.

In some embodiments the amino acid substitutions in the hIFN polypeptidemay be with naturally occurring or non-naturally occurring amino acids,provided that at least one substitution is with a non-naturally encodedamino acid.

In some embodiments, the non-naturally encoded amino acid comprises acarbonyl group, an acetyl group, an aminooxy group, a hydrazine group, ahydrazide group, a semicarbazide group, an azide group, or an alkynegroup.

In some embodiments, the non-naturally encoded amino acid comprises acarbonyl group. In some embodiments, the non-naturally encoded aminoacid has the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R₂ is H, an alkyl, aryl, substituted alkyl, andsubstituted aryl; and R₃ is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R₄ is H, an amino acid, a polypeptide,or a carboxy terminus modification group.

In some embodiments, the non-naturally encoded amino acid comprises anaminooxy group. In some embodiments, the non-naturally encoded aminoacid comprises a hydrazide group. In some embodiments, the non-naturallyencoded amino acid comprises a hydrazine group. In some embodiments, thenon-naturally encoded amino acid residue comprises a semicarbazidegroup.

In some embodiments, the non-naturally encoded amino acid residuecomprises an azide group. In some embodiments, the non-naturally encodedamino acid has the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; m is 0-10; R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, the non-naturally encoded amino acid comprises analkyne group. In some embodiments, the non-naturally encoded amino acidhas the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; X is O, N, S or not present; m is 0-10, R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, the polypeptide is a hIFN polypeptide agonist,partial agonist, antagonist, partial antagonist, or inverse agonist. Insome embodiments, the hIFN polypeptide agonist, partial agonist,antagonist, partial antagonist, or inverse agonist comprises anon-naturally encoded amino acid linked to a water soluble polymer. Insome embodiments, the water soluble polymer comprises a poly(ethyleneglycol) moiety. In some embodiments, the hIFN polypeptide agonist,partial agonist, antagonist, partial antagonist, or inverse agonistcomprises a non-naturally encoded amino acid and one or morepost-translational modification, linker, polymer, or biologically activemolecule. In some embodiments, the non-naturally encoded amino acidlinked to a water soluble polymer is present within the Site II region(the region of the protein encompassing the AC helical-bundle face,amino terminal region of helix A and a portion of helix C) of the hIFNpolypeptide. In some embodiments, the hIFN polypeptide comprising anon-naturally encoded amino acid linked to a water soluble polymerprevents dimerization of the hIFN polypeptide receptor by preventing thehIFN polypeptide antagonist from binding to a second hIFN polypeptidereceptor molecule.

The present invention also provides isolated nucleic acids comprising apolynucleotide that hybridizes under stringent conditions to SEQ ID NO:21 or 22 wherein the polynucleotide comprises at least one selectorcodon. In some embodiments, the selector codon is selected from thegroup consisting of an amber codon, ochre codon, opal codon, a uniquecodon, a rare codon, a five-base codon, and a four-base codon.

The present invention also provides methods of making a hIFN polypeptidelinked to a water soluble polymer. In some embodiments, the methodcomprises contacting an isolated hIFN polypeptide comprising anon-naturally encoded amino acid with a water soluble polymer comprisinga moiety that reacts with the non-naturally encoded amino acid. In someembodiments, the non-naturally encoded amino acid incorporated into thehIFN polypeptide is reactive toward a water soluble polymer that isotherwise unreactive toward any of the 20 common amino acids. In someembodiments, the non-naturally encoded amino acid incorporated into thehIFN polypeptide is reactive toward a linker, polymer, or biologicallyactive molecule that is otherwise unreactive toward any of the 20 commonamino acids.

In some embodiments, the hIFN polypeptide linked to the water solublepolymer is made by reacting a hIFN polypeptide comprising acarbonyl-containing amino acid with a poly(ethylene glycol) moleculecomprising an aminooxy, hydrazine, hydrazide or semicarbazide group. Insome embodiments, the aminooxy, hydrazine, hydrazide or semicarbazidegroup is linked to the poly(ethylene glycol) molecule through an amidelinkage.

In some embodiments, the hIFN polypeptide linked to the water solublepolymer is made by reacting a poly(ethylene glycol) molecule comprisinga carbonyl group with a polypeptide comprising a non-naturally encodedamino acid that comprises an aminooxy, hydrazine, hydrazide orsemicarbazide group.

In some embodiments, the hIFN polypeptide linked to the water solublepolymer is made by reacting a hIFN polypeptide comprising analkyne-containing amino acid with a poly(ethylene glycol) moleculecomprising an azide moiety. In some embodiments, the azide or alkynegroup is linked to the poly(ethylene glycol) molecule through an amidelinkage.

In some embodiments, the hIFN polypeptide linked to the water solublepolymer is made by reacting a hIFN polypeptide comprising anazide-containing amino acid with a poly(ethylene glycol) moleculecomprising an alkyne moiety. In some embodiments, the azide or alkynegroup is linked to the poly(ethylene glycol) molecule through an amidelinkage.

In some embodiments, the poly(ethylene glycol) molecule has a molecularweight of between about 0.1 and about 100 kDa. In some embodiments, thepoly(ethylene glycol) molecule has a molecular weight of between 0.1 kDaand 50 kDa.

In some embodiments, the poly(ethylene glycol) molecule is a branchedpolymer. In some embodiments, each branch of the poly(ethylene glycol)branched polymer has a molecular weight of between 1 kDa and 100 kDa, orbetween 1 kDa and 50 kDa.

In some embodiments, the water soluble polymer linked to the hIFNpolypeptide comprises a polyalkylene glycol moiety. In some embodiments,the non-naturally encoded amino acid residue incorporated into the hIFNpolypeptide comprises a carbonyl group, an aminooxy group, a hydrazidegroup, a hydrazine, a semicarbazide group, an azide group, or an alkynegroup. In some embodiments, the non-naturally encoded amino acid residueincorporated into the hIFN polypeptide comprises a carbonyl moiety andthe water soluble polymer comprises an aminooxy, hydrazide, hydrazine,or semicarbazide moiety. In some embodiments, the non-naturally encodedamino acid residue incorporated into the hIFN polypeptide comprises analkyne moiety and the water soluble polymer comprises an azide moiety.In some embodiments, the non-naturally encoded amino acid residueincorporated into the hIFN polypeptide comprises an azide moiety and thewater soluble polymer comprises an alkyne moiety.

The present invention also provides compositions comprising a hIFNpolypeptide comprising a non-naturally encoded amino acid and apharmaceutically acceptable carrier. In some embodiments, thenon-naturally encoded amino acid is linked to a water soluble polymer.

The present invention also provides cells comprising a polynucleotideencoding the hIFN polypeptide comprising a selector codon. In someembodiments, the cells comprise an orthogonal RNA synthetase and/or anorthogonal tRNA for substituting a non-naturally encoded amino acid intothe hIFN polypeptide.

The present invention also provides methods of making a hIFN polypeptidecomprising a non-naturally encoded amino acid. In some embodiments, themethods comprise culturing cells comprising a polynucleotide orpolynucleotides encoding a hIFN polypeptide, an orthogonal RNAsynthetase and/or an orthogonal tRNA under conditions to permitexpression of the hIFN polypeptide; and purifying the hIFN polypeptidefrom the cells and/or culture medium.

The present invention also provides methods of increasing therapeutichalf-life, serum half-life or circulation time of hIFN polypeptides. Thepresent invention also provides methods of modulating immunogenicity ofhIFN polypeptides. The present invention also provides methods ofmodulating toxicity of hIFN polypeptides. The present invention alsoprovides methods of modulating side effects of current IFN therapeutics.In some embodiments, the methods comprise substituting a non-naturallyencoded amino acid for any one or more amino acids in naturallyoccurring hIFN polypeptides and/or linking the hIFN polypeptide to alinker, a polymer, a water soluble polymer, or a biologically activemolecule.

The present invention also provides methods of treating a patient inneed of such treatment with an effective amount of a hIFN molecule ofthe present invention. In some embodiments, the methods compriseadministering to the patient a therapeutically-effective amount of apharmaceutical composition comprising a hIFN polypeptide comprising anon-naturally-encoded amino acid and a pharmaceutically acceptablecarrier. In some embodiments, the non-naturally encoded amino acid islinked to a water soluble polymer.

The present invention also provides hIFN polypeptides comprising asequence shown in SEQ ID NO: 1, 2, 3 or any other hIFN polypeptidesequence or interferon-like cytokine such as limitin, except that atleast one amino acid is substituted by a non-naturally encoded aminoacid. In some embodiments, the non-naturally encoded amino acid islinked to a water soluble polymer. In some embodiments, the watersoluble polymer comprises a poly(ethylene glycol) moiety. In someembodiments, the non-naturally encoded amino acid comprises a carbonylgroup, an aminooxy group, a hydrazide group, a hydrazine group, asemicarbazide group, an azide group, or an alkyne group.

The present invention also provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a hIFN polypeptidecomprising the sequence shown in SEQ ID NO: 1, 2, 3 or any other IFNpolypeptide sequence or interferon-like cytokine such as limitin,wherein at least one amino acid is substituted by a non-naturallyencoded amino acid. In some embodiments, the non-naturally encoded aminoacid comprises a saccharide moiety. In some embodiments, the watersoluble polymer is linked to the polypeptide via a saccharide moiety. Insome embodiments, a linker, polymer, or biologically active molecule islinked to the hIFN polypeptide via a saccharide moiety.

The present invention also provides a hIFN polypeptide comprising awater soluble polymer linked by a covalent bond to the hIFN polypeptideat a single amino acid. In some embodiments, the water soluble polymercomprises a poly(ethylene glycol) moiety. In some embodiments, the aminoacid covalently linked to the water soluble polymer is a non-naturallyencoded amino acid present in the polypeptide.

The present invention provides a hIFN polypeptide comprising at leastone linker, polymer, or biologically active molecule, wherein saidlinker, polymer, or biologically active molecule is attached to thepolypeptide through a functional group of a non-naturally encoded aminoacid ribosomally incorporated into the polypeptide. In some embodiments,the polypeptide is monoPEGylated. The present invention also provides ahIFN polypeptide comprising a linker, polymer, or biologically activemolecule that is attached to one or more non-naturally encoded aminoacid wherein said non-naturally encoded amino acid is ribosomallyincorporated into the polypeptide at pre-selected sites.

In another embodiment, conjugation of the hIFN polypeptide comprisingone or more non-naturally occurring amino acids to another molecule,including but not limited to PEG, provides substantially purified hIFNdue to the unique chemical reaction utilized for conjugation to thenon-natural amino acid. Conjugation of hIFN comprising one or morenon-naturally encoded amino acids to another molecule, such as PEG, maybe performed with other purification techniques performed prior to orfollowing the conjugation step to provide substantially pure hIFN.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—A diagram of the general structure for the four helical bundleprotein Interferon alpha-2 (IFNα-2) is shown.

FIG. 2—A diagram of results obtained from the pSTAT1 assay is shown.

FIG. 3—A diagram showing the specificity of the pSTAT1 assay is shown.

FIG. 4—A diagram of the effect of endotoxin on the pSTAT1 assay isshown.

FIG. 5—A diagram of results obtained from an anti-proliferation assay isshown.

FIG. 6—A diagram of the effect of endotoxin on the anti-proliferationassay is shown.

FIG. 7—A diagram of results obtained from an assay measuring inductionof HLA expression is shown.

FIG. 8—A diagram of Tyk2 phosphorylation in U266 cells is shown.

FIG. 9—A diagram of CPE results obtained with nine PEGylated hIFNpolypeptides and PEGASYS® is shown.

FIG. 10—A graph of CFU-GM colony count vs. International Units is shownfor PEGASYS® and four hIFN polypeptides.

FIG. 11—An analysis of anti-viral IC50 and K_(D) for IFNR2 is shown.

FIG. 12—Results from a hematopoietic toxicity assay are shown.

FIG. 13—Results from a hematopoietic toxicity assay are shown.

FIG. 14—Results from a hematopoietic toxicity assay are shown.

FIG. 15—Results from a hematopoietic toxicity assay are shown.

FIG. 16—Results from a hematopoietic toxicity assay are shown.

FIG. 17—Results from a hematopoietic toxicity assay are shown.

FIG. 18—A diagram of CPE results obtained with five PEGylated hIFNpolypeptides and PEGASYS®.

FIG. 19—A diagram is shown of the structure of linear, 30 kDamonomethoxy-poly(ethylene glycol)-2-aminooxy ethylamine carbamatehydrochloride.

FIG. 20—A diagram is shown of the synthesis of mPEG (40K)aminoethoxyamine hydrochloride from mPEG (40K) p-nitrophenolcarbonate.

DEFINITIONS

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, constructs, and reagentsdescribed herein and as such may vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention, which will be limited only by the appended claims.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. Thus, for example, reference to a “hIFN” is a reference toone or more such proteins and includes equivalents thereof known tothose of ordinary skill in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devices,and materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications and patents mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, theconstructs and methodologies that are described in the publications,which might be used in connection with the presently describedinvention. The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the inventors arenot entitled to antedate such disclosure by virtue of prior invention orfor any other reason.

The term “substantially purified” refers to a hIFN polypeptide that maybe substantially or essentially free of components that normallyaccompany or interact with the protein as found in its naturallyoccurring environment, i.e. a native cell, or host cell in the case ofrecombinantly produced hIFN polypeptides. hIFN polypeptide that may besubstantially free of cellular material includes preparations of proteinhaving less than about 30%, less than about 25%, less than about 20%,less than about 15%, less than about 10%, less than about 5%, less thanabout 4%, less than about 3%, less than about 2%, or less than about 1%(by dry weight) of contaminating protein. When the hIFN polypeptide orvariant thereof is recombinantly produced by the host cells, the proteinmay be present at about 30%, about 25%, about 20%, about 15%, about 10%,about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dryweight of the cells. When the hIFN polypeptide or variant thereof isrecombinantly produced by the host cells, the protein may be present inthe culture medium at about 5 g/L, about 4 g/L, about 3 g/L, about 2g/L, about 1 g/L, about 750 mg/L, about 500 mg/L, about 250 mg/L, about100 mg/L, about 50 mg/L, about 10 mg/L, or about 1 mg/L or less of thedry weight of the cells. Thus, “substantially purified” hIFN polypeptideas produced by the methods of the present invention may have a puritylevel of at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, specifically, a puritylevel of at least about 75%, 80%, 85%, and more specifically, a puritylevel of at least about 90%, a purity level of at least about 95%, apurity level of at least about 99% or greater as determined byappropriate methods such as SDS/PAGE analysis, RP-HPLC, SEC, andcapillary electrophoresis.

A “recombinant host cell” or “host cell” refers to a cell that includesan exogenous polynucleotide, regardless of the method used forinsertion, for example, direct uptake, transduction, f-mating, or othermethods known in the art to create recombinant host cells. The exogenouspolynucleotide may be maintained as a nonintegrated vector, for example,a plasmid, or alternatively, may be integrated into the host genome.

As used herein, the term “medium” or “media” includes any culturemedium, solution, solid, semi-solid, or rigid support that may supportor contain any host cell, including bacterial host cells, yeast hostcells, insect host cells, plant host cells, eukaryotic host cells,mammalian host cells, CHO cells, prokaryotic host cells, E. coli, orPseudomonas host cells, and cell contents. Thus, the term may encompassmedium in which the host cell has been grown, e.g., medium into whichthe hIFN polypeptide has been secreted, including medium either beforeor after a proliferation step. The term also may encompass buffers orreagents that contain host cell lysates, such as in the case where thehIFN polypeptide is produced intracellularly and the host cells arelysed or disrupted to release the hIFN polypeptide.

“Reducing agent,” as used herein with respect to protein refolding, isdefined as any compound or material which maintains sulfhydryl groups inthe reduced state and reduces intra- or intermolecular disulfide bonds.Suitable reducing agents include, but are not limited to, dithiothreitol(DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine(2-aminoethanethiol), and reduced glutathione. It is readily apparent tothose of ordinary skill in the art that a wide variety of reducingagents are suitable for use in the methods and compositions of thepresent invention.

“Oxidizing agent,” as used hereinwith respect to protein refolding, isdefined as any compound or material which is capable of removing anelectron from a compound being oxidized. Suitable oxidizing agentsinclude, but are not limited to, oxidized glutathione, cystine,cystamine, oxidized dithiothreitol, oxidized erythreitol, and oxygen. Itis readily apparent to those of ordinary skill in the art that a widevariety of oxidizing agents are suitable for use in the methods of thepresent invention.

“Denaturing agent” or “denaturant,” as used herein, is defined as anycompound or material which will cause a reversible unfolding of aprotein. The strength of a denaturing agent or denaturant will bedetermined both by the properties and the concentration of theparticular denaturing agent or denaturant. Suitable denaturing agents ordenaturants may be chaotropes, detergents, organic solvents, watermiscible solvents, phospholipids, or a combination of two or more suchagents. Suitable chaotropes include, but are not limited to, urea,guanidine, and sodium thiocyanate. Useful detergents may include, butare not limited to, strong detergents such as sodium dodecyl sulfate, orpolyoxyethylene ethers (e.g. Tween or Triton detergents), Sarkosyl, mildnon-ionic detergents (e.g., digitonin), mild cationic detergents such asN->2,3-(Dioleyoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents(e.g. sodium cholate or sodium deoxycholate) or zwitterionic detergentsincluding, but not limited to, sulfobetaines (Zwittergent),3-(3-chlolamidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-1-propane sulfonate(CHAPSO). Organic, water miscible solvents such as acetonitrile, loweralkanols (especially C₂-C₄ alkanols such as ethanol or isopropanol), orlower alkandiols (especially C₂-C₄ alkandiols such as ethylene-glycol)may be used as denaturants. Phospholipids useful in the presentinvention may be naturally occurring phospholipids such asphosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, andphosphatidylinositol or synthetic phospholipid derivatives or variantssuch as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

“Refolding,” as used herein describes any process, reaction or methodwhich transforms disulfide bond containing polypeptides from animproperly folded or unfolded state to a native or properly foldedconformation with respect to disulfide bonds.

“Cofolding,” as used herein, refers specifically to refolding processes,reactions, or methods which employ at least two polypeptides whichinteract with each other and result in the transformation of unfolded orimproperly folded polypeptides to native, properly folded polypeptides.

As used herein, “interferon” or “IFN” shall include those polypeptidesand proteins that have at least one biological activity of aninterferon, including but not limited to IFNα, IFNβ, IFNγ, IFNγ, IFNω,or IFNτ or interferon-like cytokines such as limitin (such as thosedescribed in U.S. Pat. Nos. 4,414,150; 4,456,748; 4,727,138; 4,762,791,4,929,554; 5,096,705; 4,695,623; 4,614,651; 4,678,751; 4,925,793;5,460,811; 5,120,832; 4,780,530; 4,908,432; 4,970,161; 4,973,479;4,975,276; 5,098,703; 5,278,286; 5,661,009; 6,372,206; 6,433,144;6,472,512; 6,572,853; 6,703,225; 6,200,780; 6,299,869; 6,300,475;6,323,006; 6,350,589; 5,705,363; 5,738,845; 5,789,551; 6,117,423;6,174,996; 5,540,923; 5,541,293; 5,541,312; 5,554,513; 5,593,667 whichare incorporated by reference herein), as well as IFN analogs, IFNisoforms, IFN mimetics, IFN fragments, hybrid IFN proteins, fusionproteins, oligomers and multimers, homologues, glycosylation patternvariants, variants, splice variants, and muteins, thereof, regardless ofthe biological activity of same, and further regardless of the method ofsynthesis or manufacture thereof including, but not limited to,recombinant (whether produced from cDNA, genomic DNA, synthetic DNA orother form of nucleic acid), in vitro, in vivo, by microinjection ofnucleic acid molecules, synthetic, transgenic, and gene activatedmethods. Specific examples of IFN include, but are not limited to,IFNγ-1b (Actimmune™), IFNβ-1a (Avonex®, and Rebif®), IFNβ-1b(Betaseron®), consensus IFN, IFN alfacon-1 (Infergen®), IFNα-2 (IntronA®), IFNα-2a (Roferon-A®), Peginterferon alfa-2a (PEGASYS®),Peginterferon alfa-2b (PEG-Intron®), IFN analog, IFN mutants, alteredglycosylated human IFN, and PEG conjugated IFN analogs. Specificexamples of cells modified for expression of endogenous human IFN aredescribed in Devlin et al., J. Leukoc. Biol. 41:306 (1987); U.S. Pat.Nos. 6,610,830; 6,482,613; 6,489,144; 6,159,712; 5,814,485; 5,710,027;5,595,888; 4,966,843; which are incorporated by reference herein. Seealso, U.S. Pat. Nos. 6,716,606; 6,379,661; 6,004,548; 5,830,705;5,582,823; 4,810,643; and 6,242,218, which are incorporated by referenceherein, for expression of GH family members.

The term “human IFN (hIFN)” or “hIFN polypeptide” refers to interferonor IFN as described above, as well as a polypeptide that retains atleast one biological activity of a naturally-occurring hIFN. The term“hIFN polypeptides” or “hIFN” also includes the pharmaceuticallyacceptable salts and prodrugs, and prodrugs of the salts, polymorphs,hydrates, solvates, biologically-active fragments, biologically-activevariants and stereoisomers of the naturally-occurring human IFN as wellas agonist, mimetic, and antagonist variants of the naturally-occurringhuman IFN and polypeptide fusions thereof. Examples of hIFN polypeptidesinclude, but are not limited to, those described in U.S. Pat. No.4,604,284; 5,582,824; 6,531,122; 6,204,022; 6,120,762; 6,046,034;6,036,956; 5,939,286; 5,908,626; 5,780,027; 5,770,191; 5,723,125;5,594,107; 5,378,823; 4,898,931; 4,892,743, which are incorporated byreference herein. Fusions comprising additional amino acids at the aminoterminus, carboxyl terminus, or both, are encompassed by the term “hIFNpolypeptide.” Exemplary fusions include, but are not limited to, e.g.,methionyl IFN in which a methionine is linked to the N-terminus of hIFNresulting from the recombinant expression of the mature form of hIFNlacking the secretion signal peptide or portion thereof, fusions for thepurpose of purification (including but not limited to, to poly-histidineor affinity epitopes), fusions with serum albumin binding peptides andfusions with serum proteins such as serum albumin. Also the term “hIFNpolypeptide” also includes hybrid molecules that have one or more aminoacid substitutions from limitin or other interferon-like cytokines. U.S.Pat. No. 5,750,373, which is incorporated by reference herein, describesa method for selecting novel proteins such as growth hormone andantibody fragment variants having altered binding properties for theirrespective receptor molecules. The method comprises fusing a geneencoding a protein of interest to the carboxy terminal domain of thegene III coat protein of the filamentous phage M13. Thenaturally-occurring hIFN nucleic acid and amino acid sequences forfull-length and mature forms are known, as are variants such as singleamino acid variants or splice variants.

Consensus interferon is a recombinant type 1 interferon containing 166amino acids. Consensus IFN was derived by scanning the sequences ofseveral natural alpha interferons and assigning the most frequentlyobserved amino acid in each corresponding position. Consensus IFN, whencompared on an equal mass basis with IFNα-2a and α-2b in in vitroassays, typically displays 5-10 times higher biological activity (Blattet al. J. Interferon Cytokine Res. 1996; 16:489-99).

Modified hIFN polypeptides may exhibit one or more properties orbiological activities found with a different interferon molecule. Forexample, a hIFN polypeptide that was generated from an IFNα-2a aminoacid sequence and that comprises one or more non-naturally encoded aminoacid that is unPEGylated or PEGylated may exhibit one or more biologicalactivity that is found with IFNβ. One such activity may beanti-proliferative activity.

For the complete full-length naturally-occurring IFNα-2a amino acidsequence as well as the mature naturally-occurring IFNα-2a amino acidsequence, see SEQ ID NO: 1, and SEQ ID NO: 2, respectively, herein. Insome embodiments, hIFN polypeptides of the invention are substantiallyidentical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or any othersequence of an interferon polypeptide or interferon-like cytokine suchas limitin. Nucleic acid molecules encoding hIFN mutants and mutant hIFNpolypeptides are well known and include, but are not limited to, thosedisclosed in U.S. Pat. Nos. 6,331,525; 6,069,133; 5,955,307; 5,869,293;5,831,062; 5,081,022; 5,004,689; 4,738,931; 4,686,191; which areincorporated by reference herein. Examples of hIFN mutants include thosedisclosed in U.S. Pat. Nos. 6,514,729 and 5,582,824, which areincorporated by reference herein.

Interferons have a variety of biological activities, includinganti-viral, immunoregulatory and anti-proliferative properties, and havebeen utilized as therapeutic agents for treatment of diseases such ascancer, and various viral diseases. Interferon-α's have been shown toinhibit various types of cellular proliferation, and are especiallyuseful for the treatment of a variety of cellular proliferationdisorders frequently associated with cancer, particularly hematologicmalignancies such as leukemias. These proteins have shownanti-proliferative activity against multiple myeloma, chroniclymphocytic leukemia, low-grade lymphoma, Kaposi's sarcoma, chronicmyelogenous leukemia, renal-cell carcinoma, urinary bladder tumors andovarian cancers (Bonnem, E. M. et al. (1984) J. Biol. Response Modifiers3:580; Oldham, R. K. (1985) Hospital Practice 20:71).

IFNα's are useful against various types of viral infections (Finter, N.B. et al. (1991) Drugs 42(5):749). Interferon-α's have shown activityagainst human papillomavirus infection, Hepatitis B, and Hepatitis Cinfections (Finter, N. B. et al., 1991, supra; Kashima, H. et al. (1988)Laryngoscope 98:334; Dusheiko, G. M. et al. (1986) J. Hematology 3(Supple. 2):S199; Davis, G L et al. (1989) N. England J. Med. 321:1501).The role of interferons and interferon receptors in the pathogenesis ofcertain autoimmune and inflammatory diseases has also been investigated(Benoit, P. et al. (1993) J. Immunol. 150(3):707). In addition,interferon-α has been approved for use for the treatment of diseasessuch as hairy cell leukemia, renal cell carcinoma, basal cell carcinoma,malignant melanoma, AIDS-related Kaposi's sarcoma, multiple myeloma,chronic myelogenous leukemia, non-Hodgkin's lymphoma, laryngealpapillomatosis, mycosis fungoides, condyloma acuminata, chronichepatitis B, hepatitis C, chronic hepatitis D, and chronic non-A,non-B/C hepatitis.

Interferons have been implicated in the pathogenesis of variousautoimmune diseases, such as systemic lupus erythematoses, Behcet'sdisease, and insulin-dependent diabetes mellitus (IDDM, also referred toas type I diabetes). It has been demonstrated in a transgenic mousemodel that β cell expression of IFN-αcan cause insulitis and IDDM, andIFN-αantagonists (including antibodies) have been proposed for thetreatment of IDDM (WO 93/04699, published Mar. 18, 1993). Impaired IFN-γand IFN-αproduction has been observed in multiple sclerosis (MS)patients. IFN-αhas been detected in the serum of many AIDS patients, andit has been reported that the production of IFN-γ is greatly suppressedin suspensions of mitogen-stimulated mononuclear cells derived from AIDSpatients. For a review see, for example, Chapter 16, “The Presence andPossible Pathogenic Role of Interferons in Disease”, In: Interferons andother Regulatory Cytokines, Edward de Maeyer (1988, John Wiley and Sonspublishers). Alpha and beta interferons have been used in the treatmentof the acute viral disease herpes zoster (T. C. Merigan et al, N. Engl.J. Med. 298, 981-987 (1978); E. Heidemann et al., Onkologie 7, 210-212(1984)), chronic viral infections, e.g. hepatitis C and hepatitis Binfections (R. L. Knobler et al., Neurology 34(10): 1273-9 (1984); M. A.Faerkkilae et al., Act. Neurol. Sci. 69, 184-185 (1985)). rIFNα-2a(Roferon®, Roche) is an injection formulation indicated in use for thetreatment of hairy cell leukemia and AIDS-related Kaposi's sarcoma.Recombinant IFNα-2b (Intron A®, Schering) has been approved for thetreatment of hairy cell leukemia, selected cases of condylomataacuminata, AIDS-related Kaposi's sarcoma, chronic hepatitis C, andchronic hepatitis B infections in certain patients. Compositions ofmultiple subtypes of IFNα are also used to treat a variety of diseases(Multiferon®, Viragen, Inc.). IFNγ1b (Actimmune®, IntermunePharmaceuticals, Inc.) is commercially available for the treatment ofchronic granulomatous disease and malignant osteopetrosis.

The biologic activities of type I IFNs have been disclosed and are knownin the art, and can be found, for example, in Pfeffer, Semin. Oncol. 24(suppl 9), S9-63-S9-69 (1997) and U.S. Pat. Nos. 6,436,391; 6,372,218;6,270,756; 6,207,145; 6,086,869; 6,036,949; 6,013,253; 6,007,805;5,980,884; 5,958,402; 5,863,530; 5,849,282; 5,846,526; 5,830,456;5,824,300; 5,817,307; 5,780,021; 5,624,895; 5,480,640; 5,268,169;5,208,019; 5,196,191; 5,190,751; 5,104,653; 5,019,382; 4,959,210; whichare incorporated by reference herein. A related application is U.S.patent application entitled “Modified Human Interferon Polypeptides andTheir Uses” published as US 2005/0220762 on Oct. 6, 2005, which isincorporated by reference herein.

IFNα's are members of the diverse helical-bundle superfamily of cytokinegenes (Sprang, S. R. et al. (1993) Curr. Opin. Struct. Biol. 3:815-827).The human interferon a's are encoded by a family of over 20 tandemlyduplicated nonallelic genes that share 85-98% sequence identity at theamino acid level (Henco, K. et al. (1985) J. Mol. Biol. 185:227-260).Human IFNβ is a regulatory polypeptide with a molecular weight of about22 kDa consisting of 166 amino acid residues. It can be produced by mostcells in the body, in particular fibroblasts, in response to viralinfection or exposure to other agents. It binds to a multimeric cellsurface receptor, and productive receptor binding results in a cascadeof intracellular events leading to the expression of IFNβ induciblegenes which in turn produces effects which can be classified asanti-viral, anti-proliferative and immunomodulatory.

The amino acid sequence of human IFNβ is known and was reported forexample by Taniguchi, Gene 10:11-15, 1980, and in EP 83069, EP 41313 andU.S. Pat. No. 4,686,191 which are incorporated by reference herein.Crystal structures have been reported for human and murine IFNβ,respectively (Proc. Natl. Acad. Sci. USA 94:11813-11818, 1997; J. Mol.Biol. 253:187-207, 1995; U.S. Pat. Nos. 5,602,232; 5,460,956; 5,441,734;4,672,108; which are incorporated by reference herein). They have beenreviewed in Cell Mol. Life. Sci. 54:1203-1206, 1998. Variants of IFNβhave been reported (WO 95/25170, U.S. Pat. No. 6,572,853, U.S. Pat. No.5,545,723, U.S. Pat. No. 4,914,033, EP 260350, U.S. Pat. No. 4,588,585,U.S. Pat. No. 4,769,233, Stewart et al, DNA Vol. 6 no. 2 1987 pp.119-128, Runkel et al, 1998, J. Biol. Chem. 273, No. 14, pp. 8003-8008,which are incorporated by reference herein). Expression of IFNβ in CHOcells has been reported (U.S. Pat. No. 4,966,843, U.S. Pat. No.5,376,567 and U.S. Pat. No. 5,795,779, which are incorporated byreference herein). IFNβ molecules with a particular glycosylationpattern and methods for their preparation have been reported (EP 287075and EP 529300).

Commercial preparations of IFNβ are sold under the names Betaseron®(also termed interferon β1b, which is non-glycosylated, produced usingrecombinant bacterial cells, has a deletion of the N-terminal methionineresidue and the C17S mutation), and Avonex® and Rebif® (also termedinterferon β1a, which is glycosylated, produced using recombinantmammalian cells) for treatment of patients with multiple sclerosis, haveshown to be effective in reducing the exacerbation rate, and morepatients remain exacerbation-free for prolonged periods of time ascompared with placebo-treated patients. Furthermore, the accumulationrate of disability is reduced (Neurol. 51:682-689, 1998).

Comparison of IFNβ1a and β1b with respect to structure and function hasbeen presented in Pharmaceut. Res. 15:641-649, 1998. IFNβ has been shownto delay the progression of multiple sclerosis, a relapsing thenprogressive inflammatory degenerative disease of the central nervoussystem. IFNβ may have inhibitory effects on the proliferation ofleukocytes and antigen presentation. IFNβ may modulate the profile ofcytokine production towards an anti-inflammatory phenotype. IFNβ canreduce T-cell migration by inhibiting the activity of T-cell matrixmetalloproteases. These activities are likely to act in concert toaccount for the mechanism of IFNβ in MS (Neurol. 51:682-689, 1998).

IFNβ may be used for the treatment of osteosarcoma, basal cellcarcinoma, cervical dysplasia, glioma, acute myeloid leukemia, multiplemyeloma, Hodgkin's disease, breast carcinoma, melanoma, and viralinfections such as papilloma virus, viral hepatitis, herpes genitalis,herpes zoster, herpetic keratitis, herpes simplex, viral encephalitis,cytomegalovirus pneumonia, and rhinovirus, Various side effects areassociated with the use of current preparations of IFNβ, includinginjection site reactions, fever, chills, myalgias, arthralgias, andother flu-like symptoms (Clin. Therapeutics, 19:883-893, 1997).

Given the multitude of side effects with current IFNβ products, theirassociation with frequent injection, the risk of developing neutralizingantibodies impeding the desired therapeutic effect of IFNβ, and thepotential for obtaining more optimal therapeutic IFNβ levels withconcomitant enhanced therapeutic effect, there is clearly a need forimproved IFNβ-like molecules.

Additional side effects found in patients receiving interferon therapyinclude flu-like symptoms such as fatigue, headache, and fever,anorexia, myelosuppression, and neutropenia. Side effects such as thesecan require cessation of treatment or a reduction of dosage. SeeJonasch, E. Oncologist 2001; 6:34-55, which is incorporated byreference. IFNα2a therapy has other associated toxicities includingthrombocytopenia. Among the most common side effects of IFNα currenttherapy are pyrexia, headache, rigors, and myalgia. Side effects thatlead to dose reductions are most commonly hematological consequences ofbone marrow suppression, including anemia and neutropenia. Adverseeffects of IFNs are problematic even with IFN/ribavirin combinationtherapy; the combined use of a PEGylated interferonα (PEGASYS® orPEG-Intron®) and Ribavirin (Copegus®) is the current standard of care inHCV therapy. Moreover, patients with HCV genotype 1 (1a or 1b) have beenshown to be much less likely to respond to combination therapy than HCVgenotype 2 or 3. Also, these combination regimens are found to beremarkably less effective in patients with a high viral load. Thus,there is clearly a need for improved IFN-like therapeutics.

Other interferons (IFN-ε, IFN-κ, IFN-δ, IFN-τ) and four interferon-likecytokines (limitin, IL-28A, IL-28B, IL-29) have been described. Theinteraction of interferons and interferon-like molecules with receptormolecules as well as their biological activities and clinicalapplications are described by Pestka, S. et al. in “Interferons,interferon-like cytokines, and their receptors,” Immunol Rev. 2004December; 202:8-32, which is incorporated by reference herein. Type Ireceptor chains IFNαR1 and IFNαR2 are utilized by a number of theinterferons including IFN-α, IFN-β, and limitin. The gene encodingsequence of IFNαR1 was originally cloned by Uze et al. Cell 1990;60:225, which is incorporated by reference herein. It is a 110 kDaprotein, whereas IFNαR2 occurs in two different forms. The two forms ofIFNαR2 are from the same gene and are generated as differentiallyspliced products (Lutfalla et al. EMBO J. 1995; 14:5100; Domanski et al.J. Biol. Chem. 1995; 270:21606; and Novick et al. Cell 1994; 77:391,which are incorporated by reference herein). The short form (IFNAR2b)has a molecular mass of 51 kDa and the long form (IFNAR2c) is 90-100kDa. Thus, two forms of the receptor complex thus exist, with IFNαR1associating with IFNAR2b or IFNAR2c. Colamonici et al. J. Biol. Chem.1994; 269:5660, which is incorporated by reference herein, have shownthat both types of receptor transduce signals and mediate the biologicaleffects of interferons. IFNs utilize the Jak-Stat signal transductionpathway in addition to other pathways.

Limitin (Interferon-zeta) has been found only in mice and was isolatedbased on its ability to inhibit the proliferation of a myelomonocyticleukemia cell line (Oritani, K. et al. Nature Medicine 20006(6):659-666, which is incorporated herein by reference). Limitin (SEQID NO: 23) has been shown to kill or arrest the proliferation of certainlympho-hematopoietic cell lines (B lymphopoiesis), but showed littleinfluence on erythropoiesis or myelopoiesis. Sequence analysis of the182 amino acid protein has shown homology to IFN-α and IFN-β (31.9% and25.9% identical in 166 overlapping amino acids) and a hydrophobic set ofresidues at the amino terminus. Kawamoto et al. J. Virol. 2003September; 77(17):9622-31, which is incorporated by reference, describestudies of the antiviral activity of limitin with encephalomyocarditisvirus (EMCV) and herpes simplex virus (HSV) infected cells, and plaqueformation in mouse hepatitis virus (MHV) infected cells.

Differences between limitin and interferons such as IFNα have beendiscussed, including, but not limited to, differences in signaltransduction pathways for antiviral effects, in the antiviral effectsthemselves, and in myelosuppressive effects. See Kawamoto et al. J.Virol. 2003 September; 77(17):9622-31. Kawamoto et al. ExperimentalHematology 2004; 32:797-805, which is incorporated by reference herein,describes differences found between limitin and IFN-α in assaysmeasuring antiviral, immunomodulatory, antitumor, and myelosuppressiveactivity and in an in vivo study. Limitin was found to separateanti-viral activity from bone marrow toxicity.

Thirteen different alpha interferons transmit distinct signals through asingle paired x/p two chain receptor complex. Modulating of componentsinvolved in one or more signaling pathways mediated through the samereceptor complex may provide optimization of the antiviral effects ofhIFN polypeptides, and modulation of toxic side effects. A number ofmolecules are involved with downstream signaling pathways. Antisenseoligonucleotides to CrkL and CrkII that inhibited protein expression ofthese molecules were used by Platanias et al. and were found to reversethe inhibition of IFNα or IFNγ on proliferation of bone marrow cells(CFU-GM and BFU-E). IFNα is known to activate the STAT1 pathway, andknockout mouse studies point to the importance of the STAT1 signalingcascade in fighting viral infections. Durbin et al. (Cell. 1996 Feb. 9;84(3):443-50) have shown that mice homozygous for a STAT1 knockoutdevelop spontaneous viral infections unless they are raised in a sterileenvironment. In addition, signaling through STAT5 and CrkL ultimatelylead to activation of the RAP1 protein which may be necessary forproducing growth-inhibitory signals. The phosphorylation of STAT3 mayalso be measured.

Various references disclose modification of polypeptides by polymerconjugation or glycosylation. The term “hIFN polypeptide” includespolypeptides conjugated to a polymer such as PEG and may be comprised ofone or more additional derivitizations of cysteine, lysine, or otherresidues. In addition, the hIFN polypeptide may comprise a linker orpolymer, wherein the amino acid to which the linker or polymer isconjugated may be a non-natural amino acid according to the presentinvention, or may be conjugated to a naturally encoded amino acidutilizing techniques known in the art such as coupling to lysine orcysteine.

Polymer modification of native IFNβ or a C17S variant thereof has beenreported (EP 229108, U.S. Pat. No. 5,382,657; EP 593868; U.S. Pat. No.4,917,888 and WO 99/55377, which are incorporated by reference herein).U.S. Pat. No. 4,904,584 discloses PEGylated lysine depletedpolypeptides, wherein at least one lysine residue has been deleted orreplaced with any other amino acid residue. WO 99/67291 discloses aprocess for conjugating a protein with PEG, wherein at least one aminoacid residue on the protein is deleted and the protein is contacted withPEG under conditions sufficient to achieve conjugation to the protein.WO 99/03887 discloses PEGylated variants of polypeptides belonging tothe growth hormone superfamily, wherein a cysteine residue has beensubstituted with a non-essential amino acid residue located in aspecified region of the polypeptide. Examples of PEGylated IFN moleculesinclude those disclosed in U.S. Pat. Nos. 6,524,570; 6,250,469;6,180,096; 6,177,074; 6,042,822; 5,981,709; 5,951,974; 5,908,621;5,738,846; 5,711,944; 5,382,657, which are incorporated by referenceherein. IFNβ is mentioned as one example of a polypeptide belonging tothe growth hormone superfamily. WO 00/23114 discloses glycosylated andpegylated IFNβ. WO 00/23472 discloses IFNβ fusion proteins. WO 00/26354discloses a method of producing a glycosylated polypeptide variant withreduced allergenicity, which as compared to a corresponding parentpolypeptide comprises at least one additional glycosylation site. IFNβis disclosed as one example among many polypeptides that can be modifiedaccording to the technology described in U.S. Pat. No. 5,218,092, whichis incorporated by reference herein. U.S. Pat. No. 5,218,092, which isincorporated by reference herein, discloses modification of granulocytecolony stimulating factor (G-CSF) and other polypeptides so as tointroduce at least one additional carbohydrate chain as compared to thenative polypeptide.

The term “hIFN polypeptide” also includes glycosylated hIFN, such as butnot limited to, polypeptides glycosylated at any amino acid position,N-linked or O-linked glycosylated forms of the polypeptide. These formsincluded, but are not limited to, a polypeptide with an O-linkedglycosylation site at position 129 of SEQ ID NO: 1, or the equivalentposition of SEQ ID NO: 2 or 3, or any other IFN polypeptide (Adolf etal., Biochem. J. 276:511 (1991)).

Variants containing single nucleotide changes are also considered asbiologically active variants of hIFN polypeptide. In addition, splicevariants are also included. The term “hIFN polypeptide” also includeshIFN polypeptide heterodimers, homodimers, heteromultimers, orhomomultimers of any one or more hIFN polypeptides or any otherpolypeptide, protein, carbohydrate, polymer, small molecule, linker,ligand, or other biologically active molecule of any type, linked bychemical means or expressed as a fusion protein, as well as polypeptideanalogues containing, for example, specific deletions or othermodifications yet maintain biological activity.

All references to amino acid positions in hIFN described herein arebased on the position in SEQ ID NO: 2, unless otherwise specified (i.e.,when it is stated that the comparison is based on SEQ ID NO: 1, 3, orother hIFN sequence or interferon-like cytokine such as limitin). Thoseof skill in the art will appreciate that amino acid positionscorresponding to positions in SEQ ID NO: 1, 2, 3, or any other IFNsequence or interferon-like cytokine such as limitin can be readilyidentified in any other hIFN molecule or interferon-like cytokine suchas limitin such as hIFN fusions, variants, fragments, etc. For example,sequence alignment programs such as BLAST can be used to align andidentify a particular position in a protein that corresponds with aposition in SEQ ID NO: 1, 2, 3, or other IFN sequence or interferon-likecytokine such as limitin. Substitutions, deletions or additions of aminoacids described herein in reference to SEQ ID NO: 1, 2, 3, or other IFNsequence are intended to also refer to substitutions, deletions oradditions in corresponding positions in hIFN fusions, variants,fragments, interferon-like cytokines such as limitin, etc. describedherein or known in the art and are expressly encompassed by the presentinvention.

The term “hIFN polypeptide” or “hIFN” encompasses hIFN polypeptidescomprising one or more amino acid substitutions, additions or deletions.hIFN polypeptides of the present invention may be comprised ofmodifications with one or more natural amino acids in conjunction withone or more non-natural amino acid modification. For example, a hIFNpolypeptide may comprise a non-naturally encoded amino acid substitutionas well as a natural amino acid substitution for the first amino acid atthe N-terminus. Exemplary substitutions in a wide variety of amino acidpositions in naturally-occurring hIFN polypeptides have been described,including but not limited to substitutions that modulate one or more ofthe biological activities of the hIFN polypeptide, such as but notlimited to, increase agonist activity, increase solubility of thepolypeptide, decrease protease susceptibility, convert the polypeptideinto an antagonist, etc. and are encompassed by the term “hIFNpolypeptide.”

Human IFN antagonists include, but are not limited to, those withsubstitutions at: 2, 3, 4, 5, 7, 8, 16, 19, 20, 40, 42, 50, 51, 58, 68,69, 70, 71, 73, 97, 105, 109, 112, 118, 148, 149, 152, 153, 158, 163,164, 165, or any combination thereof (SEQ ID NO: 2, or the correspondingamino acid in SEQ ID NO: 1, 3, or any other IFN sequence); a hIFNpolypeptide comprising one of these substitutions may potentially act asa weak antagonist or weak agonist depending on the site selected and thedesired activity. Human IFN antagonists include, but are not limited to,those with substitutions at 22, 23, 24, 25, 26, 27, 28, 30, 31, 32, 33,34, 35, 74, 77, 78, 79, 80, 82, 83, 85, 86, 89, 90, 93, 94, 124, 125,127, 128, 129, 131, 132, 133, 134, 135, 136, 137, or any combinationthereof (hIFN; SEQ ID NO: 2 or the corresponding amino acids in SEQ IDNO: 1 or 3). In some embodiments, hIFN antagonists comprise at least onesubstitution in the regions 1-9 (N-terminus), 10-21 (A helix), 22-39(region between A helix and B helix), 40-75 (B helix), 76-77 (regionbetween B helix and C helix), 78-100 (C helix), 101-110 (region betweenC helix and D helix), 111-132 (D helix), 133-136 (region between D and Ehelix), 137-155 (E helix), 156-165 (C-terminus) that cause IFN to act asan antagonist. In other embodiments, the exemplary sites ofincorporation of a non-naturally encoded amino acid include residueswithin the amino terminal region of helix A and a portion of helix C. Inother embodiments, the above-listed substitutions are combined withadditional substitutions that cause the hIFN polypeptide to be a hIFNantagonist. In some embodiments, the hIFN antagonist comprises anon-naturally encoded amino acid linked to a water soluble polymer thatis present in a receptor binding region of the hIFN molecule.

In some embodiments, the hIFN polypeptides further comprise an addition,substitution or deletion that modulates biological activity of the hIFNpolypeptide. For example, the additions, substitutions or deletions maymodulate one or more properties or activities of hIFN. For example, theadditions, substitutions or deletions may modulate affinity for the hIFNpolypeptide receptor, modulate (including but not limited to, increasesor decreases) receptor dimerization, modulate downstream signalingevents post hIFN receptor binding, stabilize receptor dimers, modulatecirculating half-life, modulate therapeutic half-life, modulatestability of the polypeptide, modulate cleavage by proteases, modulatedose, modulate release or bio-availability, modulate one or more sideeffects found with current IFN therapeutics, modulate toxicity,facilitate purification, or improve or alter a particular route ofadministration. Similarly, hIFN polypeptides may comprise proteasecleavage sequences, reactive groups, antibody-binding domains (includingbut not limited to, FLAG or poly-His) or other affinity based sequences(including but not limited to, FLAG, poly-His, GST, etc.) or linkedmolecules (including but not limited to, biotin) that improve detection(including but not limited to, GFP), purification or other traits of thepolypeptide.

The term “hIFN polypeptide” also encompasses homodimers, heterodimers,homomultimers, and heteromultimers that are linked, including but notlimited to those linked directly via non-naturally encoded amino acidside chains, either to the same or different non-naturally encoded aminoacid side chains, to naturally-encoded amino acid side chains, orindirectly via a linker. Exemplary linkers including but are not limitedto, small organic compounds, water soluble polymers of a variety oflengths such as poly(ethylene glycol) or polydextran, or polypeptides ofvarious lengths.

A “non-naturally encoded amino acid” refers to an amino acid that is notone of the 20 common amino acids or pyrrolysine or selenocysteine. Otherterms that may be used synonymously with the term “non-naturally encodedamino acid” are “non-natural amino acid,” “unnatural amino acid,”“non-naturally-occurring amino acid,” and variously hyphenated andnon-hyphenated versions thereof. The term “non-naturally encoded aminoacid” also includes, but is not limited to, amino acids that occur bymodification (e.g. post-translational modifications) of a naturallyencoded amino acid (including but not limited to, the 20 common aminoacids or pyrrolysine and selenocysteine) but are not themselvesnaturally incorporated into a growing polypeptide chain by thetranslation complex. Examples of such non-naturally-occurring aminoacids include, but are not limited to, N-acetylglucosaminyl-L-serine,N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

An “amino terminus modification group” refers to any molecule that canbe attached to the amino terminus of a polypeptide. Similarly, a“carboxy terminus modification group” refers to any molecule that can beattached to the carboxy terminus of a polypeptide. Terminus modificationgroups include, but are not limited to, various water soluble polymers,peptides or proteins such as serum albumin, or other moieties thatincrease serum half-life of peptides.

The terms “functional group”, “active moiety”, “activating group”,“leaving group”, “reactive site”, “chemically reactive group” and“chemically reactive moiety” are used in the art and herein to refer todistinct, definable portions or units of a molecule. The terms aresomewhat synonymous in the chemical arts and are used herein to indicatethe portions of molecules that perform some function or activity and arereactive with other molecules.

The term “linkage” or “linker” is used herein to refer to groups orbonds that normally are formed as the result of a chemical reaction andtypically are covalent linkages. Hydrolytically stable linkages meansthat the linkages are substantially stable in water and do not reactwith water at useful pH values, including but not limited to, underphysiological conditions for an extended period of time, perhaps evenindefinitely. Hydrolytically unstable or degradable linkages mean thatthe linkages are degradable in water or in aqueous solutions, includingfor example, blood. Enzymatically unstable or degradable linkages meanthat the linkage can be degraded by one or more enzymes. As understoodin the art, PEG and related polymers may include degradable linkages inthe polymer backbone or in the linker group between the polymer backboneand one or more of the terminal functional groups of the polymermolecule. For example, ester linkages formed by the reaction of PEGcarboxylic acids or activated PEG carboxylic acids with alcohol groupson a biologically active agent generally hydrolyze under physiologicalconditions to release the agent. Other hydrolytically degradablelinkages include, but are not limited to, carbonate linkages; iminelinkages resulted from reaction of an amine and an aldehyde; phosphateester linkages formed by reacting an alcohol with a phosphate group;hydrazone linkages which are reaction product of a hydrazide and analdehyde; acetal linkages that are the reaction product of an aldehydeand an alcohol; orthoester linkages that are the reaction product of aformate and an alcohol; peptide linkages formed by an amine group,including but not limited to, at an end of a polymer such as PEG, and acarboxyl group of a peptide; and oligonucleotide linkages formed by aphosphoramidite group, including but not limited to, at the end of apolymer, and a 5′ hydroxyl group of an oligonucleotide.

The term “biologically active molecule”, “biologically active moiety” or“biologically active agent” when used herein means any substance whichcan affect any physical or biochemical properties of a biologicalsystem, pathway, molecule, or interaction relating to an organism,including but not limited to, viruses, bacteria, bacteriophage,transposon, prion, insects, fungi, plants, animals, and humans. Inparticular, as used herein, biologically active molecules include, butare not limited to, any substance intended for diagnosis, cure,mitigation, treatment, or prevention of disease in humans or otheranimals, or to otherwise enhance physical or mental well-being of humansor animals. Examples of biologically active molecules include, but arenot limited to, peptides, proteins, enzymes, small molecule drugs, harddrugs, soft drugs, carbohydrates, inorganic atoms or molecules, dyes,lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells,viruses, liposomes, microparticles and micelles. Classes of biologicallyactive agents that are suitable for use with the invention include, butare not limited to, drugs, prodrugs, radionuclides, imaging agents,polymers, antibiotics, fungicides, anti-viral agents, anti-inflammatoryagents, anti-tumor agents, cardiovascular agents, anti-anxiety agents,hormones, growth factors, steroidal agents, microbially derived toxins,and the like.

A “bifunctional polymer” refers to a polymer comprising two discretefunctional groups that are capable of reacting specifically with othermoieties (including but not limited to, amino acid side groups) to formcovalent or non-covalent linkages. A bifunctional linker having onefunctional group reactive with a group on a particular biologicallyactive component, and another group reactive with a group on a secondbiological component, may be used to form a conjugate that includes thefirst biologically active component, the bifunctional linker and thesecond biologically active component. Many procedures and linkermolecules for attachment of various compounds to peptides are known.See, e.g., European Patent Application No. 188,256; U.S. Pat. Nos.4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; and 4,569,789which are incorporated by reference herein. A “multi-functional polymer”refers to a polymer comprising two or more discrete functional groupsthat are capable of reacting specifically with other moieties (includingbut not limited to, amino acid side groups) to form covalent ornon-covalent linkages. A bi-functional polymer or multi-functionalpolymer may be any desired length or molecular weight, and may beselected to provide a particular desired spacing or conformation betweenone or more molecules linked to hIFN and its receptor or hIFN.

Where substituent groups are specified by their conventional chemicalformulas, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, for example, the structure —CH₂O— isequivalent to the structure —OCH₂—.

The term “substituents” includes but is not limited to “non-interferingsubstituents”. “Non-interfering substituents” are those groups thatyield stable compounds. Suitable non-interfering substituents orradicals include, but are not limited to, halo, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ alkoxy, C₁-C₁₂ aralkyl, C₁-C₁₂ alkaryl,C₃-C₁₂ cycloalkyl, C₃-C₁₂ cycloalkenyl, phenyl, substituted phenyl,toluoyl, xylenyl, biphenyl, C₂-C₁₂ alkoxyalkyl, C₂-C₁₂ alkoxyaryl,C₇-C₁₂ aryloxyalkyl, C₇-C₁₂ oxyaryl, C₁-C₆ alkylsulfinyl, C₁-C₁₀alkylsulfonyl, —(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein m is from 1 to 8,aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclicradical, substituted heterocyclic radical, nitroalkyl, —NO₂, —CN,—NRC(O)—(C₁-C₁₀ alkyl), —C(O)—(C₁-C₁₀ alkyl), C₂-C₁₀ alkyl thioalkyl,—C(O)O—(C₁-C₁₀ alkyl), —OH, —SO₂, ═S, —COOH, —NR₂, carbonyl,—C(O)—(C₁-C₁₀ alkyl)-CF₃, —C(O)—CF₃, —C(O)NR₂, —(C₁-C₁₀ aryl)-S—(C₆-C₁₀aryl), —C(O)—(C₁-C₁₀ aryl), —(CH₂)_(m)—O—(—(CH₂)_(m)—O—(C₁-C₁₀ alkyl)wherein each m is from 1 to 8, —C(O)NR₂, —C(S)NR₂, —SO₂NR₂, —NRC(O)NR₂,—NRC(S)NR₂, salts thereof, and the like. Each R as used herein is H,alkyl or substituted alkyl, aryl or substituted aryl, aralkyl, oralkaryl.

The term “halogen” includes fluorine, chlorine, iodine, and bromine.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups whichare limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by the structures —CH₂CH₂— and —CH₂CH₂CH₂CH₂—, and furtherincludes those groups described below as “heteroalkylene.” Typically, analkyl (or alkylene) group will have from 1 to 24 carbon atoms, withthose groups having 10 or fewer carbon atoms being a particularembodiment of the methods and compositions described herein. A “loweralkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂, —S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may beconsecutive, such as, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.Similarly, the term “heteroalkylene” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, the same or differentheteroatoms can also occupy either or both of the chain termini(including but not limited to, alkyleneoxy, alkylenedioxy,alkyleneamino, alkylenediamino, aminooxyalkylene, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both —C(O)₂R′— and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Thus, a cycloalkylor heterocycloalkyl include saturated, partially unsaturated and fullyunsaturated ring linkages. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl,3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkylinclude, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl),1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,2-piperazinyl, and the like. Additionally, the term encompasses bicyclicand tricyclic ring structures. Similarly, the term “heterocycloalkylene”by itself or as part of another substituent means a divalent radicalderived from heterocycloalkyl, and the term “cycloalkylene” by itself oras part of another substituent means a divalent radical derived fromcycloalkyl.

As used herein, the term “water soluble polymer” refers to any polymerthat is soluble in aqueous solvents. Linkage of water soluble polymersto hIFN polypeptides can result in changes including, but not limitedto, increased or modulated serum half-life, or increased or modulatedtherapeutic half-life relative to the unmodified form, modulatedimmunogenicity, modulated physical association characteristics such asaggregation and multimer formation, altered receptor binding, alteredreceptor dimerization or multimerization, modulated toxicity, andmodulation of one or more the biological activities of IFN includingside effects found with current IFN therapeutics. The water solublepolymer may or may not have its own biological activity, and may beutilized as a linker for attaching hIFN to other substances, includingbut not limited to one or more hIFN polypeptides, or one or morebiologically active molecules. Suitable polymers include, but are notlimited to, polyethylene glycol, polyethylene glycol propionaldehyde,mono C1-C10 alkoxy or aryloxy derivatives thereof (described in U.S.Pat. No. 5,252,714 which is incorporated by reference herein),monomethoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinylalcohol, polyamino acids, divinylether maleic anhydride,N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivativesincluding dextran sulfate, polypropylene glycol, polypropyleneoxide/ethylene oxide copolymer, polyoxyethylated polyol, heparin,heparin fragments, polysaccharides, oligosaccharides, glycans, celluloseand cellulose derivatives, including but not limited to methylcelluloseand carboxymethyl cellulose, starch and starch derivatives,polypeptides, polyalkylene glycol and derivatives thereof, copolymers ofpolyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers,and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, ormixtures thereof. Examples of such water soluble polymers include, butare not limited to, polyethylene glycol and serum albumin.

As used herein, the term “polyalkylene glycol” or “poly(alkene glycol)”refers to polyethylene glycol (poly(ethylene glycol)), polypropyleneglycol, polybutylene glycol, and derivatives thereof. The term“polyalkylene glycol” encompasses both linear and branched polymers andaverage molecular weights of between 0.1 kDa and 100 kDa. Otherexemplary embodiments are listed, for example, in commercial suppliercatalogs, such as Shearwater Corporation's catalog “Polyethylene Glycoland Derivatives for Biomedical Applications” (2001).

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (including but not limited to from 1 to 3 rings) which are fusedtogether or linked covalently. The term “heteroaryl” refers to arylgroups (or rings) that contain from one to four heteroatoms selectedfrom N, O, and S, wherein the nitrogen and sulfur atoms are optionallyoxidized, and the nitrogen atom(s) are optionally quaternized. Aheteroaryl group can be attached to the remainder of the moleculethrough a heteroatom. Non-limiting examples of aryl and heteroarylgroups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl,pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl,3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl,purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituentsfor each of the above noted aryl and heteroaryl ring systems areselected from the group of acceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(including but not limited to, aryloxy, arylthioxy, arylalkyl) includesboth aryl and heteroaryl rings as defined above. Thus, the term“arylalkyl” is meant to include those radicals in which an aryl group isattached to an alkyl group (including but not limited to, benzyl,phenethyl, pyridylmethyl and the like) including those alkyl groups inwhich a carbon atom (including but not limited to, a methylene group)has been replaced by, for example, an oxygen atom (including but notlimited to, phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl,and the like).

Each of the above terms (including but not limited to, “alkyl,”“heteroalkyl,” “aryl” and “heteroaryl”) are meant to include bothsubstituted and unsubstituted forms of the indicated radical. Exemplarysubstituents for each type of radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″))═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such a radical. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, but are not limited to: halogen, —OR′, ═O, ═NR′, ═N—OR′,—NR′R″, —SR, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, andfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number ofopen valences on the aromatic ring system; and where R′, R″, R′″ and R″″are independently selected from hydrogen, alkyl, heteroalkyl, aryl andheteroaryl. When a compound of the invention includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′″ and R″″ groups when more than one of these groupsis present.

As used herein, the term “modulated serum half-life” means the positiveor negative change in circulating half-life of a modified hIFN relativeto its non-modified form. Serum half-life is measured by taking bloodsamples at various time points after administration of hIFN, anddetermining the concentration of that molecule in each sample.Correlation of the serum concentration with time allows calculation ofthe serum half-life. Increased serum half-life desirably has at leastabout two-fold, but a smaller increase may be useful, for example whereit enables a satisfactory dosing regimen or avoids a toxic effect. Insome embodiments, the increase is at least about three-fold, at leastabout five-fold, or at least about ten-fold.

The term “modulated therapeutic half-life” as used herein means thepositive or negative change in the half-life of the therapeuticallyeffective amount of hIFN, relative to its non-modified form. Therapeutichalf-life is measured by measuring pharmacokinetic and/orpharmacodynamic properties of the molecule at various time points afteradministration. Increased therapeutic half-life desirably enables aparticular beneficial dosing regimen, a particular beneficial totaldose, or avoids an undesired effect. In some embodiments, the increasedtherapeutic half-life results from increased potency, increased ordecreased binding of the modified molecule to its target, increased ordecreased breakdown on the molecule by enzymes such as proteases, or anincrease or decrease in another parameter or mechanism of action of thenon-modified molecule.

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is free of at least some of thecellular components with which it is associated in the natural state, orthat the nucleic acid or protein has been concentrated to a levelgreater than the concentration of its in vivo or in vitro production. Itcan be in a homogeneous state. Isolated substances can be in either adry or semi-dry state, or in solution, including but not limited to, anaqueous solution. It can be a component of a pharmaceutical compositionthat comprises additional pharmaceutically acceptable carriers and/orexcipients. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames which flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to substantially one band in anelectrophoretic gel. Particularly, it may mean that the nucleic acid orprotein is at least 85% pure, at least 90% pure, at least 95% pure, atleast 99% or greater pure.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers to oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-naturally encoded amino acid. As used herein, the terms encompassamino acid chains of any length, including full length proteins, whereinthe amino acid residues are linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and pyrrolysine and selenocysteine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of ordinary skill inthe art will recognize that each codon in a nucleic acid (except AUG,which is ordinarily the only codon for methionine, and TGG, which isordinarily the only codon for tryptophan) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

As to amino acid sequences, one of ordinary skill in the art willrecognize that individual substitutions, deletions or additions to anucleic acid, peptide, polypeptide, or protein sequence which alters,adds or deletes a single amino acid or a small percentage of amino acidsin the encoded sequence is a “conservatively modified variant” where thealteration results in the deletion of an amino acid, addition of anamino acid, or substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are known to those of ordinary skill in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention.

Conservative substitution tables providing functionally similar aminoacids are known to those of ordinary skill in the art. The followingeight groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd edition (December 1993)

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Sequences are“substantially identical” if they have a percentage of amino acidresidues or nucleotides that are the same (i.e., about 60% identity,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, orabout 95% identity over a specified region), when compared and alignedfor maximum correspondence over a comparison window, or designatedregion as measured using one of the following sequence comparisonalgorithms (or other algorithms available to persons of ordinary skillin the art) or by manual alignment and visual inspection. Thisdefinition also refers to the complement of a test sequence. Theidentity can exist over a region that is at least about 50 amino acidsor nucleotides in length, or over a region that is 75-100 amino acids ornucleotides in length, or, where not specified, across the entiresequence of a polynucleotide or polypeptide.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are known to those of ordinary skill in the art. Optimalalignment of sequences for comparison can be conducted, including butnot limited to, by the local homology algorithm of Smith and Waterman(1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci.USA 85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Ausubel et al., CurrentProtocols in Molecular Biology (1995 supplement)).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1997) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Informationavailable at the World Wide Web at ncbi.nlm.nih.gov. The BLAST algorithmparameters W, T, and X determine the sensitivity and speed of thealignment. The BLASTN program (for nucleotide sequences) uses asdefaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4 anda comparison of both strands. For amino acid sequences, the BLASTPprogram uses as defaults a wordlength of 3, and expectation (E) of 10,and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of10, M=5, N=−4, and a comparison of both strands. The BLAST algorithm istypically performed with the “low complexity” filter turned off.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, or less than about0.01, or less than about 0.001.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (including but not limited to,total cellular or library DNA or RNA).

The phrase “stringent hybridization conditions” refers to hybridizationof sequences of DNA, RNA, PNA, or other nucleic acid mimics, orcombinations thereof under conditions of low ionic strength and hightemperature as is known in the art. Typically, under stringentconditions a probe will hybridize to its target subsequence in a complexmixture of nucleic acid (including but not limited to, total cellular orlibrary DNA or RNA) but does not hybridize to other sequences in thecomplex mixture. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Probes, “Overview of principles of hybridization and thestrategy of nucleic acid assays” (1993). Generally, stringent conditionsare selected to be about 5-10° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength pH. TheT_(m) is the temperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions may be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (including butnot limited to, 10 to 50 nucleotides) and at least about 60° C. for longprobes (including but not limited to, greater than 50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. For selective or specifichybridization, a positive signal may be at least two times background,optionally 10 times background hybridization. Exemplary stringenthybridization conditions can be as following: 50% formamide, 5×SSC, and1% SDS, incubating at 42° C., or 5×SSC, 1% SDS, incubating at 65° C.,with wash in 0.2×SSC, and 0.1% SDS at 65° C. Such washes can beperformed for 5, 15, 30, 60, 120, or more minutes.

As used herein, the term “eukaryote” refers to organisms belonging tothe phylogenetic domain Eucarya such as animals (including but notlimited to, mammals, insects, reptiles, birds, etc.), ciliates, plants(including but not limited to, monocots, dicots, algae, etc.), fungi,yeasts, flagellates, microsporidia, protists, etc.

As used herein, the term “non-eukaryote” refers to non-eukaryoticorganisms. For example, a non-eukaryotic organism can belong to theEubacteria (including but not limited to, Escherichia coli, Thermusthermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens,Pseudomonas aeruginosa, Pseudomonas putida, etc.) phylogenetic domain,or the Archaea (including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium such as Haloferaxvolcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus,Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.)phylogenetic domain.

The term “subject” as used herein, refers to an animal, in someembodiments a mammal, and in other embodiments a human, who is theobject of treatment, observation or experiment.

The term “effective amount” as used herein refers to that amount of themodified non-natural amino acid polypeptide being administered whichwill relieve to some extent one or more of the symptoms of the disease,condition or disorder being treated. Compositions containing themodified non-natural amino acid polypeptide described herein can beadministered for prophylactic, enhancing, and/or therapeutic treatments.

The terms “enhance” or “enhancing” means to increase or prolong eitherin potency or duration a desired effect. Thus, in regard to enhancingthe effect of therapeutic agents, the term “enhancing” refers to theability to increase or prolong, either in potency or duration, theeffect of other therapeutic agents on a system. An “enhancing-effectiveamount,” as used herein, refers to an amount adequate to enhance theeffect of another therapeutic agent in a desired system. When used in apatient, amounts effective for this use will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician.

The term “modified,” as used herein refers to any changes made to agiven polypeptide, such as changes to the length of the polypeptide, theamino acid sequence, chemical structure, co-translational modification,or post-translational modification of a polypeptide. The form“(modified)” term means that the polypeptides being discussed areoptionally modified, that is, the polypeptides under discussion can bemodified or unmodified.

The term “post-translationally modified” refers to any modification of anatural or non-natural amino acid that occurs to such an amino acidafter it has been incorporated into a polypeptide chain. The termencompasses, by way of example only, co-translational in vivomodifications, co-translational in vitro modifications (such as in acell-free translation system), post-translational in vivo modifications,and post-translational in vitro modifications.

In prophylactic applications, compositions containing the modifiednon-natural amino acid polypeptide are administered to a patientsusceptible to or otherwise at risk of a particular disease, disorder orcondition. Such an amount is defined to be a “prophylactically effectiveamount.” In this use, the precise amounts also depend on the patient'sstate of health, weight, and the like. It is considered well within theskill of the art for one to determine such prophylactically effectiveamounts by routine experimentation (e.g., a dose escalation clinicaltrial).

The term “protected” refers to the presence of a “protecting group” ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin or with the methods and compositions described herein, includingphotolabile groups such as Nvoc and MeNvoc. Other protecting groupsknown in the art may also be used in or with the methods andcompositions described herein.

By way of example only, blocking/protecting groups may be selected from:

Other protecting groups are described in Greene and Wuts, ProtectiveGroups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, N.Y.,1999, which is incorporated herein by reference in its entirety.

In therapeutic applications, compositions containing the modifiednon-natural amino acid polypeptide are administered to a patient alreadysuffering from a disease, condition or disorder, in an amount sufficientto cure or at least partially arrest the symptoms of the disease,disorder or condition. Such an amount is defined to be a“therapeutically effective amount,” and will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician. It is considered well within the skill of theart for one to determine such therapeutically effective amounts byroutine experimentation (e.g., a dose escalation clinical trial).

The term “treating” is used to refer to either prophylactic and/ortherapeutic treatments.

Non-naturally encoded amino acid polypeptides presented herein mayinclude isotopically-labelled compounds with one or more atoms replacedby an atom having an atomic mass or mass number different from theatomic mass or mass number usually found in nature. Examples of isotopesthat can be incorporated into the present compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as ²H,³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ¹⁸F, ³⁶Cl, respectively. Certainisotopically-labelled compounds described herein, for example those intowhich radioactive isotopes such as ³H and ¹⁴C are incorporated, may beuseful in drug and/or substrate tissue distribution assays. Further,substitution with isotopes such as deuterium, i.e., 2H, can affordcertain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements.

All isomers including but not limited to diastereomers, enantiomers, andmixtures thereof are considered as part of the compositions describedherein. In additional or further embodiments, the non-naturally encodedamino acid polypeptides are metabolized upon administration to anorganism in need to produce a metabolite that is then used to produce adesired effect, including a desired therapeutic effect. In further oradditional embodiments are active metabolites of non-naturally encodedamino acid polypeptides.

In some situations, non-naturally encoded amino acid polypeptides mayexist as tautomers. In addition, the non-naturally encoded amino acidpolypeptides described herein can exist in unsolvated as well assolvated forms with pharmaceutically acceptable solvents such as water,ethanol, and the like. The solvated forms are also considered to bedisclosed herein. Those of ordinary skill in the art will recognize thatsome of the compounds herein can exist in several tautomeric forms. Allsuch tautomeric forms are considered as part of the compositionsdescribed herein.

Unless otherwise indicated, conventional methods of mass spectroscopy,NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniquesand pharmacology, within the skill of the art are employed.

DETAILED DESCRIPTION

I. Introduction

Interferon molecules comprising at least one unnatural amino acid areprovided in the invention. In certain embodiments of the invention, thehIFN polypeptide with at least one unnatural amino acid includes atleast one post-translational modification. In one embodiment, the atleast one post-translational modification comprises attachment of amolecule including but not limited to, a label, a dye, a polymer, awater-soluble polymer, a derivative of polyethylene glycol, aphotocrosslinker, a radionuclide, a cytotoxic compound, a drug, anaffinity label, a photoaffinity label, a reactive compound, a resin, asecond protein or polypeptide or polypeptide analog, an antibody orantibody fragment, a metal chelator, a cofactor, a fatty acid, acarbohydrate, a polynucleotide, a DNA, a RNA, an antisensepolynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin,an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spinlabel, a fluorophore, a metal-containing moiety, a radioactive moiety, anovel functional group, a group that covalently or noncovalentlyinteracts with other molecules, a photocaged moiety, an actinicradiation excitable moiety, a photoisomerizable moiety, biotin, aderivative of biotin, a biotin analogue, a moiety incorporating a heavyatom, a chemically cleavable group, a photocleavable group, an elongatedside chain, a carbon-linked sugar, a redox-active agent, an aminothioacid, a toxic moiety, an isotopically labeled moiety, a biophysicalprobe, a phosphorescent group, a chemiluminescent group, an electrondense group, a magnetic group, an intercalating group, a chromophore, anenergy transfer agent, a biologically active agent, a detectable label,a small molecule, a quantum dot, a nanotransmitter, a radionucleotide, aradiotransmitter, a neutron-capture agent, or any combination of theabove or any other desirable compound or substance, comprising a secondreactive group to at least one unnatural amino acid comprising a firstreactive group utilizing chemistry methodology that is known to one ofordinary skill in the art to be suitable for the particular reactivegroups. For example, the first reactive group is an alkynyl moiety(including but not limited to, in the unnatural amino acidp-propargyloxyphenylalanine, where the propargyl group is also sometimesreferred to as an acetylene moiety) and the second reactive group is anazido moiety, and [3+2]cycloaddition chemistry methodologies areutilized. In another example, the first reactive group is the azidomoiety (including but not limited to, in the unnatural amino acidp-azido-L-phenylalanine) and the second reactive group is the alkynylmoiety. In certain embodiments of the modified hIFN polypeptide of thepresent invention, at least one unnatural amino acid (including but notlimited to, unnatural amino acid containing a keto functional group)comprising at least one post-translational modification, is used wherethe at least one post-translational modification comprises a saccharidemoiety. In certain embodiments, the post-translational modification ismade in vivo in a eukaryotic cell or in a non-eukaryotic cell. A linker,polymer, water soluble polymer, or other molecule may attach themolecule to the polypeptide. The molecule may be linked directly to thepolypeptide.

In certain embodiments, the protein includes at least onepost-translational modification that is made in vivo by one host cell,where the post-translational modification is not normally made byanother host cell type. In certain embodiments, the protein includes atleast one post-translational modification that is made in vivo by aeukaryotic cell, where the post-translational modification is notnormally made by a non-eukaryotic cell. Examples of post-translationalmodifications include, but are not limited to, glycosylation,acetylation, acylation, lipid-modification, palmitoylation, palmitateaddition, phosphorylation, glycolipid-linkage modification, and thelike. In one embodiment, the post-translational modification comprisesattachment of an oligosaccharide to an asparagine by a GlcNAc-asparaginelinkage (including but not limited to, where the oligosaccharidecomprises (GlcNAc-Man)₂-Man-GlcNAc-GlcNAc, and the like). In anotherembodiment, the post-translational modification comprises attachment ofan oligosaccharide (including but not limited to, Gal-GalNAc,Gal-GlcNAc, etc.) to a serine or threonine by a GalNAc-serine, aGalNAc-threonine, a GlcNAc-serine, or a GlcNAc-threonine linkage. Incertain embodiments, a protein or polypeptide of the invention cancomprise a secretion or localization sequence, an epitope tag, a FLAGtag, a polyhistidine tag, a GST fusion, and/or the like. Examples ofsecretion signal sequences include, but are not limited to, aprokaryotic secretion signal sequence, a eukaryotic secretion signalsequence, a eukaryotic secretion signal sequence 5′-optimized forbacterial expression, a novel secretion signal sequence, pectate lyasesecretion signal sequence, Omp A secretion signal sequence, and a phagesecretion signal sequence. Examples of secretion signal sequences,include, but are not limited to, STII (prokaryotic), Fd GIII and M13(phage), Bgl2 (yeast), and the signal sequence bla derived from atransposon.

The protein or polypeptide of interest can contain at least one, atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, or ten or more unnaturalamino acids. The unnatural amino acids can be the same or different, forexample, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more differentsites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moredifferent unnatural amino acids. In certain embodiments, at least one,but fewer than all, of a particular amino acid present in a naturallyoccurring version of the protein is substituted with an unnatural aminoacid.

The present invention provides methods and compositions based on membersof the GH supergene family, in particular hIFN, comprising at least onenon-naturally encoded amino acid. Introduction of at least onenon-naturally encoded amino acid into hIFN can allow for the applicationof conjugation chemistries that involve specific chemical reactions,including, but not limited to, with one or more non-naturally encodedamino acids while not reacting with the commonly occurring 20 aminoacids. In some embodiments, hIFN comprising the non-naturally encodedamino acid is linked to a water soluble polymer, such as polyethyleneglycol (PEG), via the side chain of the non-naturally encoded aminoacid. This invention provides a highly efficient method for theselective modification of proteins with PEG derivatives, which involvesthe selective incorporation of non-genetically encoded amino acids,including but not limited to, those amino acids containing functionalgroups or substituents not found in the 20 naturally incorporated aminoacids, including but not limited to a ketone, an azide or acetylenemoiety, into proteins in response to a selector codon and the subsequentmodification of those amino acids with a suitably reactive PEGderivative. Once incorporated, the amino acid side chains can then bemodified by utilizing chemistry methodologies known to those of ordinaryskill in the art to be suitable for the particular functional groups orsubstituents present in the non-naturally encoded amino acid. Knownchemistry methodologies of a wide variety are suitable for use in thepresent invention to incorporate a water soluble polymer into theprotein. Such methodologies include but are not limited to a Huisgen[3+2]cycloaddition reaction (see, e.g., Padwa, A. in ComprehensiveOrganic Synthesis, Vol. 4, (1991) Ed. Trost, B. M., Pergamon, Oxford, p.1069-1109; and, Huisgen, R. in 1,3-Dipolar Cycloaddition Chemistry,(1984) Ed. Padwa, A., Wiley, New York, p. 1-176) with, including but notlimited to, acetylene or azide derivatives, respectively.

Because the Huisgen [3+2]cycloaddition method involves a cycloadditionrather than a nucleophilic substitution reaction, proteins can bemodified with extremely high selectivity. The reaction can be carriedout at room temperature in aqueous conditions with excellentregioselectivity (1,4>1,5) by the addition of catalytic amounts of Cu(I)salts to the reaction mixture. See, e.g., Tornoe, et al., (2002) J. Org.Chem. 67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int.Ed. 41:2596-2599; and WO 03/101972. A molecule that can be added to aprotein of the invention through a [3+2]cycloaddition includes virtuallyany molecule with a suitable functional group or substituent includingbut not limited to an azido or acetylene derivative. These molecules canbe added to an unnatural amino acid with an acetylene group, includingbut not limited to, p-propargyloxyphenylalanine, or azido group,including but not limited to p-azido-phenylalanine, respectively.

The five-membered ring that results from the Huisgen [3+2]cycloadditionis not generally reversible in reducing environments and is stableagainst hydrolysis for extended periods in aqueous environments.Consequently, the physical and chemical characteristics of a widevariety of substances can be modified under demanding aqueous conditionswith the active PEG derivatives of the present invention. Even moreimportantly, because the azide and acetylene moieties are specific forone another (and do not, for example, react with any of the 20 common,genetically-encoded amino acids), proteins can be modified in one ormore specific sites with extremely high selectivity.

The invention also provides water soluble and hydrolytically stablederivatives of PEG derivatives and related hydrophilic polymers havingone or more acetylene or azide moieties. The PEG polymer derivativesthat contain acetylene moieties are highly selective for coupling withazide moieties that have been introduced selectively into proteins inresponse to a selector codon. Similarly, PEG polymer derivatives thatcontain azide moieties are highly selective for coupling with acetylenemoieties that have been introduced selectively into proteins in responseto a selector codon.

More specifically, the azide moieties comprise, but are not limited to,alkyl azides, aryl azides and derivatives of these azides. Thederivatives of the alkyl and aryl azides can include other substituentsso long as the acetylene-specific reactivity is maintained. Theacetylene moieties comprise alkyl and aryl acetylenes and derivatives ofeach. The derivatives of the alkyl and aryl acetylenes can include othersubstituents so long as the azide-specific reactivity is maintained.

The present invention provides conjugates of substances having a widevariety of functional groups, substituents or moieties, with othersubstances including but not limited to a label; a dye; a polymer; awater-soluble polymer; a derivative of polyethylene glycol; aphotocrosslinker; a radionuclide; a cytotoxic compound; a drug; anaffinity label; a photoaffinity label; a reactive compound; a resin; asecond protein or polypeptide or polypeptide analog; an antibody orantibody fragment; a metal chelator; a cofactor; a fatty acid; acarbohydrate; a polynucleotide; a DNA; a RNA; an antisensepolynucleotide; a saccharide; a water-soluble dendrimer; a cyclodextrin;an inhibitory ribonucleic acid; a biomaterial; a nanoparticle; a spinlabel; a fluorophore, a metal-containing moiety; a radioactive moiety; anovel functional group; a group that covalently or noncovalentlyinteracts with other molecules; a photocaged moiety; an actinicradiation excitable moiety; a photoisomerizable moiety; biotin; aderivative of biotin; a biotin analogue; a moiety incorporating a heavyatom; a chemically cleavable group; a photocleavable group; an elongatedside chain; a carbon-linked sugar; a redox-active agent; an aminothioacid; a toxic moiety; an isotopically labeled moiety; a biophysicalprobe; a phosphorescent group; a chemiluminescent group; an electrondense group; a magnetic group; an intercalating group; a chromophore; anenergy transfer agent; a biologically active agent; a detectable label;a small molecule; a quantum dot; a nanotransmitter; a radionucleotide; aradiotransmitter; a neutron-capture agent; or any combination of theabove, or any other desirable compound or substance. The presentinvention also includes conjugates of substances having azide oracetylene moieties with PEG polymer derivatives having the correspondingacetylene or azide moieties. For example, a PEG polymer containing anazide moiety can be coupled to a biologically active molecule at aposition in the protein that contains a non-genetically encoded aminoacid bearing an acetylene functionality. The linkage by which the PEGand the biologically active molecule are coupled includes but is notlimited to the Huisgen [3+2]cycloaddition product.

It is well established in the art that PEG can be used to modify thesurfaces of biomaterials (see, e.g., U.S. Pat. No. 6,610,281; Mehvar,R., J. Pharm Sci., 3(1):125-136 (2000) which are incorporated byreference herein). The invention also includes biomaterials comprising asurface having one or more reactive azide or acetylene sites and one ormore of the azide- or acetylene-containing polymers of the inventioncoupled to the surface via the Huisgen [3+2]cycloaddition linkage.Biomaterials and other substances can also be coupled to the azide- oracetylene-activated polymer derivatives through a linkage other than theazide or acetylene linkage, such as through a linkage comprising acarboxylic acid, amine, alcohol or thiol moiety, to leave the azide oracetylene moiety available for subsequent reactions.

The invention includes a method of synthesizing the azide- andacetylene-containing polymers of the invention. In the case of theazide-containing PEG derivative, the azide can be bonded directly to acarbon atom of the polymer. Alternatively, the azide-containing PEGderivative can be prepared by attaching a linking agent that has theazide moiety at one terminus to a conventional activated polymer so thatthe resulting polymer has the azide moiety at its terminus. In the caseof the acetylene-containing PEG derivative, the acetylene can be bondeddirectly to a carbon atom of the polymer. Alternatively, theacetylene-containing PEG derivative can be prepared by attaching alinking agent that has the acetylene moiety at one terminus to aconventional activated polymer so that the resulting polymer has theacetylene moiety at its terminus.

More specifically, in the case of the azide-containing PEG derivative, awater soluble polymer having at least one active hydroxyl moietyundergoes a reaction to produce a substituted polymer having a morereactive moiety, such as a mesylate, tresylate, tosylate or halogenleaving group, thereon. The preparation and use of PEG derivativescontaining sulfonyl acid halides, halogen atoms and other leaving groupsare known to those of ordinary skill in the art. The resultingsubstituted polymer then undergoes a reaction to substitute for the morereactive moiety an azide moiety at the terminus of the polymer.Alternatively, a water soluble polymer having at least one activenucleophilic or electrophilic moiety undergoes a reaction with a linkingagent that has an azide at one terminus so that a covalent bond isformed between the PEG polymer and the linking agent and the azidemoiety is positioned at the terminus of the polymer. Nucleophilic andelectrophilic moieties, including amines, thiols, hydrazides,hydrazines, alcohols, carboxylates, aldehydes, ketones, thioesters andthe like, are known to those of ordinary skill in the art.

More specifically, in the case of the acetylene-containing PEGderivative, a water soluble polymer having at least one active hydroxylmoiety undergoes a reaction to displace a halogen or other activatedleaving group from a precursor that contains an acetylene moiety.Alternatively, a water soluble polymer having at least one activenucleophilic or electrophilic moiety undergoes a reaction with a linkingagent that has an acetylene at one terminus so that a covalent bond isformed between the PEG polymer and the linking agent and the acetylenemoiety is positioned at the terminus of the polymer. The use of halogenmoieties, activated leaving group, nucleophilic and electrophilicmoieties in the context of organic synthesis and the preparation and useof PEG derivatives is well established to practitioners in the art.

The invention also provides a method for the selective modification ofproteins to add other substances to the modified protein, including butnot limited to water soluble polymers such as PEG and PEG derivativescontaining an azide or acetylene moiety. The azide- andacetylene-containing PEG derivatives can be used to modify theproperties of surfaces and molecules where biocompatibility, stability,solubility and lack of immunogenicity are important, while at the sametime providing a more selective means of attaching the PEG derivativesto proteins than was previously known in the art.

II. Growth Hormone Supergene Family

The following proteins include those encoded by genes of the growthhormone (GH) supergene family (Bazan, F., Immunology Today 11: 350-354(1990); Bazan, J. F. Science 257: 410-413 (1992); Mott, H. R. andCampbell, I. D., Current Opinion in Structural Biology 5: 114-121(1995); Silvennoinen, O. and Ihle, J. N., SIGNALLING BY THEHEMATOPOIETIC CYTOKINE RECEPTORS (1996)): growth hormone, prolactin,placental lactogen, erythropoietin (EPO), thrombopoietin (TPO),interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11,IL-12 (p35 subunit), IL-13, IL-15, oncostatin M, ciliary neurotrophicfactor (CNTF), leukemia inhibitory factor (LIF), alpha interferon, betainterferon, epsilon interferon, gamma interferon, omega interferon, tauinterferon, granulocyte-colony stimulating factor (G-CSF),granulocyte-macrophage colony stimulating factor (GM-CSF), macrophagecolony stimulating factor (M-CSF) and cardiotrophin-1 (CT-1) (“the GHsupergene family”). It is anticipated that additional members of thisgene family will be identified in the future through gene cloning andsequencing. Members of the GH supergene family have similar secondaryand tertiary structures, despite the fact that they generally havelimited amino acid or DNA sequence identity. The shared structuralfeatures allow new members of the gene family to be readily identifiedand the non-natural amino acid methods and compositions described hereinsimilarly applied. Given the extent of structural homology among themembers of the GH supergene family, non-naturally encoded amino acidsmay be incorporated into any members of the GH supergene family usingthe present invention. Each member of this family of proteins comprisesa four helical bundle. The general structure of family member IFNα-2 isshown in FIG. 1.

Structures of a number of cytokines, including G-CSF (Zink et al., FEBSLett. 314:435 (1992); Zink et al., Biochemistry 33:8453 (1994); Hill etal., Proc. Natl. Acad. Sci. USA 90:5167 (1993)), GM-CSF (Diederichs, K.,et al. Science 154: 1779-1782 (1991); Walter et al., J. Mol. Biol.224:1075-1085 (1992)), IL-2 (Bazan, J. F. and McKay, D. B. Science 257:410-413 (1992)), IL-4 (Redfield et al., Biochemistry 30: 11029-11035(1991); Powers et al., Science 256:1673-1677 (1992)), and IL-5 (Milburnet al., Nature 363: 172-176 (1993)) have been determined by X-raydiffraction and NMR studies and show striking conservation with the GHstructure, despite a lack of significant primary sequence homology. IFNis considered to be a member of this family based upon modeling andother studies (Lee et al., J. Interferon Cytokine Res. 15:341 (1995);Murgolo et al., Proteins 17:62 (1993); Radhakrishnan et al., Structure4:1453 (1996); Klaus et al., J. Mol. Biol. 274:661 (1997)). EPO isconsidered to be a member of this family based upon modeling andmutagenesis studies (Boissel et al., J. Biol. Chem. 268: 15983-15993(1993); Wen et al., J. Biol. Chem. 269: 22839-22846 (1994)). All of theabove cytokines and growth factors are now considered to comprise onelarge gene family.

In addition to sharing similar secondary and tertiary structures,members of this family share the property that they must oligomerizecell surface receptors to activate intracellular signaling pathways.Some GH family members, including but not limited to; GH and EPO, bind asingle type of receptor and cause it to form homodimers. Other familymembers, including but not limited to, IL-2, IL-4, and IL-6, bind morethan one type of receptor and cause the receptors to form heterodimersor higher order aggregates (Davis et al., (1993), Science 260:1805-1808; Paonessa et al., (1995), EMBO J. 14: 1942-1951; Mott andCampbell, Current Opinion in Structural Biology 5: 114-121 (1995)).Mutagenesis studies have shown that, like GH, these other cytokines andgrowth factors contain multiple receptor binding sites, typically two,and bind their cognate receptors sequentially (Mott and Campbell,Current Opinion in Structural Biology 5: 114-121 (1995); Matthews etal., (1996) Proc. Natl. Acad. Sci. USA 93: 9471-9476). Like GH, theprimary receptor binding sites for these other family members occurprimarily in the four alpha helices and the A-B loop. The specific aminoacids in the helical bundles that participate in receptor binding differamongst the family members. Most of the cell surface receptors thatinteract with members of the GH supergene family are structurallyrelated and comprise a second large multi-gene family. See, e.g. U.S.Pat. No. 6,608,183, which is incorporated by reference herein.

A general conclusion reached from mutational studies of various membersof the GH supergene family is that the loops joining the alpha helicesgenerally tend to not be involved in receptor binding. In particular theshort B-C loop appears to be non-essential for receptor binding in most,if not all, family members. For this reason, the B-C loop may besubstituted with non-naturally encoded amino acids as described hereinin members of the GH supergene family. The A-B loop, the C-D loop (andD-E loop of interferon/IL-10-like members of the GH superfamily) mayalso be substituted with a non-naturally-occurring amino acid. Aminoacids proximal to helix A and distal to the final helix also tend not tobe involved in receptor binding and also may be sites for introducingnon-naturally-occurring amino acids. In some embodiments, anon-naturally encoded amino acid is substituted at any position within aloop structure, including but not limited to, the first 1, 2, 3, 4, 5,6, 7, or more amino acids of the A-B, B-C, C-D or D-E loop. In someembodiments, one or more non-naturally encoded amino acids aresubstituted within the last 1, 2, 3, 4, 5, 6, 7, or more amino acids ofthe A-B, B-C, C-D or D-E loop.

Certain members of the GH family, including but not limited to, EPO,IL-2, IL-3, IL-4, IL-6, G-CSF, GM-CSF, TPO, IL-10, IL-12 p35, IL-13,IL-15 and beta interferon contain N-linked and/or O-linked sugars. Theglycosylation sites in the proteins occur almost exclusively in the loopregions and not in the alpha helical bundles. Because the loop regionsgenerally are not involved in receptor binding and because they aresites for the covalent attachment of sugar groups, they may be usefulsites for introducing non-naturally-occurring amino acid substitutionsinto the proteins. Amino acids that comprise the N- and O-linkedglycosylation sites in the proteins may be sites fornon-naturally-occurring amino acid substitutions because these aminoacids are surface-exposed. Therefore, the natural protein can toleratebulky sugar groups attached to the proteins at these sites and theglycosylation sites tend to be located away from the receptor bindingsites.

Additional members of the GH supergene family are likely to bediscovered in the future. New members of the GH supergene family can beidentified through computer-aided secondary and tertiary structureanalyses of the predicted protein sequences, and by selection techniquesdesigned to identify molecules that bind to a particular target. Membersof the GH supergene family typically possess four or five amphipathichelices joined by non-helical amino acids (the loop regions). Theproteins may contain a hydrophobic signal sequence at their N-terminusto promote secretion from the cell. Such later discovered members of theGH supergene family also are included within this invention. A relatedapplication is International Patent Application entitled “Modified FourHelical Bundle Polypeptides and Their Uses” published as WO 05/074650 onAug. 18, 2005, which is incorporated by reference herein.

Thus, the description of the growth hormone supergene family is providedfor illustrative purposes and by way of example only and not as a limiton the scope of the methods, compositions, strategies and techniquesdescribed herein. Further, reference to GH and IFN polypeptides in thisapplication is intended to use the generic term as an example of anymember of the GH supergene family. Thus, it is understood that themodifications and chemistries described herein with reference to hIFNpolypeptides or protein can be equally applied to any member of the GHsupergene family, including those specifically listed herein.

III. General Recombinant Nucleic Acid Methods for Use with the Invention

In numerous embodiments of the present invention, nucleic acids encodinga hIFN polypeptide of interest will be isolated, cloned and oftenaltered using recombinant methods. Such embodiments are used, includingbut not limited to, for protein expression or during the generation ofvariants, derivatives, expression cassettes, or other sequences derivedfrom a hIFN polypeptide. In some embodiments, the sequences encoding thepolypeptides of the invention are operably linked to a heterologouspromoter. Isolation of hIFN and production of IFN in host cells aredescribed in, e.g., U.S. Pat. Nos. 6,489,144; 6,410,697; 6,159,712;5,955,307; 5,814,485; 5,710,027; 5,595,888; 5,391,713; 5,244,655;5,196,323; 5,066,786; 4,966,843; 4,894,330; 4,364,863, which areincorporated by reference herein.

A nucleotide sequence encoding a hIFN polypeptide comprising anon-naturally encoded amino acid may be synthesized on the basis of theamino acid sequence of the parent polypeptide, including but not limitedto, having the amino acid sequence shown in SEQ ID NO: 2 (hIFN), andthen changing the nucleotide sequence so as to effect introduction(i.e., incorporation or substitution) or removal (i.e., deletion orsubstitution) of the relevant amino acid residue(s). The nucleotidesequence may be conveniently modified by site-directed mutagenesis inaccordance with conventional methods. Alternatively, the nucleotidesequence may be prepared by chemical synthesis, including but notlimited to, by using an oligonucleotide synthesizer, whereinoligonucleotides are designed based on the amino acid sequence of thedesired polypeptide, and preferably selecting those codons that arefavored in the host cell in which the recombinant polypeptide will beproduced. For example, several small oligonucleotides coding forportions of the desired polypeptide may be synthesized and assembled byPCR, ligation or ligation chain reaction. See, e.g., Barany, et al.,Proc. Nail. Acad. Sci. 88: 189-193 (1991); U.S. Pat. No. 6,521,427 whichare incorporated by reference herein.

This invention utilizes routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression. ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

General texts which describe molecular biological techniques includeBerger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989(“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 1999) (“Ausubel”)). These texts describe mutagenesis, the use ofvectors, promoters and many other relevant topics related to, includingbut not limited to, the generation of genes or polynucleotides thatinclude selector codons for production of proteins that includeunnatural amino acids, orthogonal tRNAs, orthogonal synthetases, andpairs thereof.

Various types of mutagenesis are used in the invention for a variety ofpurposes, including but not limited to, to produce novel synthetases ortRNAs, to mutate tRNA molecules, to mutate polynucleotides encodingsynthetases, to produce libraries of tRNAs, to produce libraries ofsynthetases, to produce selector codons, to insert selector codons thatencode unnatural amino acids in a protein or polypeptide of interest.They include but are not limited to site-directed, random pointmutagenesis, homologous recombination, DNA shuffling or other recursivemutagenesis methods, chimeric construction, mutagenesis using uracilcontaining templates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA or the like, or any combination thereof. Additional suitablemethods include point mismatch repair, mutagenesis usingrepair-deficient host strains, restriction-selection andrestriction-purification, deletion mutagenesis, mutagenesis by totalgene synthesis, double-strand break repair, and the like. Mutagenesis,including but not limited to, involving chimeric constructs, are alsoincluded in the present invention. In one embodiment, mutagenesis can beguided by known information of the naturally occurring molecule oraltered or mutated naturally occurring molecule, including but notlimited to, sequence, sequence comparisons, physical properties,tertiary, or quaternary structure, crystal structure or the like.

The texts and examples found herein describe these procedures.Additional information is found in the following publications andreferences cited within: Ling et al., Approaches to DNA mutagenesis: anoverview, Anal Biochem. 254(2): 157-178 (1997); Dale et al.,Oligonucleotide-directed random mulagenesis using the phosphorothioatemethod, Methods Mol. Biol. 57:369-374 (1996); Smith, In vitromulagenesis, Ann. Rev. Genet. 19:423-462 (1985); Botstein & Shortle,Strategies and applications of in vitro mutagenesis, Science229:1193-1201 (1985); Carter, Site-directed mutagenesis, Biochem. J.237:1-7 (1986); Kunkel, The efficiency of oligonucleotide directedmutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin) (1987); Kunkel, Rapidand efficient site-specific mutagenesis without phenotypic selection,Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Rapid andefficient site-specific mutagenesis without phenotypic selection,Methods in Enzymol. 154, 367-382 (1987); Bass et al., Mutant Trprepressors with new DNA-binding specificities, Science 242:240-245(1988); Zoller & Smith, Oligonucleotide-directed mutagenesis usingM13-derived vectors: an efficient and general procedure for theproduction of point mutations in any DNA fragment, Nucleic Acids Res.10:6487-6500 (1982); Zoller & Smith, Oligonucleotide-directedmutagenesis of DNA fragments cloned into M13 vectors, Methods inEnzymol. 100:468-500 (1983); Zoller & Smith, Oligonucleotide-directedmutagenesis: a simple method using two oligonucleotide primers and asingle-stranded DNA template, Methods in Enzymol. 154:329-350 (1987);Taylor et al., The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA, Nucl. Acids Res. 13: 8749-8764(1985); Taylor et al., The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA, Nucl.Acids Res. 13: 8765-8785 (1985); Nakamaye & Eckstein, Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 14: 9679-9698 (1986); Sayers et al., 5′-3′ Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 16:791-802 (1988); Sayers et al., Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide, (1988) Nucl. AcidsRes. 16: 803-814; Kramer et al., The gapped duplex DNA approach tooligonucleotide-directed mutation construction, Nucl. Acids Res. 12:9441-9456 (1984); Kramer & Fritz Oligonucleotide-directed constructionof mutations via gapped duplex DNA, Methods in Enzymol. 154:350-367(1987); Kramer et al., Improved enzymatic in vitro reactions in thegapped duplex DNA approach to oligonucleotide-directed construction ofmutations, Nucl. Acids Res. 16: 7207 (1988); Fritz et al.,Oligonucleotide-directed construction of mutations: a gapped duplex DNAprocedure without enzymatic reactions in vitro, Nucl. Acids Res. 16:6987-6999 (1988); Kramer et al., Different base/base mismatches arecorrected with different efficiencies by the methyl-directed DNAmismatch-repair system of E. coli, Cell 38:879-887 (1984); Carter etal., Improved oligonucleotide site-directed mutagenesis using M13vectors, Nucl. Acids Res. 13: 4431-4443 (1985); Carter, Improvedoligonucleotide-directed mutagenesis using M13 vectors, Methods inEnzymol. 154: 382-403 (1987); Eghtedarzadeh & Henikoff, Use ofoligonucleotides to generate large deletions, Nucl. Acids Res. 14: 5115(1986); Wells et al., Importance of hydrogen-bond formation instabilizing the transition state of subtilisin, Phil. Trans. R. Soc.Lond. A 317: 415-423 (1986); Nambiar et al., Total synthesis and cloningof a gene coding for the ribonuclease S protein, Science 223: 1299-1301(1984); Sakmar and Khorana, Total synthesis and expression of a gene forthe alpha-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin), Nucl. Acids Res. 14: 6361-6372 (1988); Wells etal., Cassette mutagenesis: an efficient methodfor generation of multiplemutations at defined sites, Gene 34:315-323 (1985); Grundström et al.,Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’ genesynthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Mandecki,Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis, Proc. Natl.Acad. Sci. USA, 83:7177-7181 (1986); Arnold, Protein engineering forunusual environments, Current Opinion in Biotechnology 4:450-455 (1993);Sieber, et al., Nature Biotechnology, 19:456-460 (2001); W. P. C.Stemmer, Nature 370, 389-91 (1994); and, I. A. Lorimer, I. Pastan,Nucleic Acids Res. 23, 3067-8 (1995). Additional details on many of theabove methods can be found in Methods in Enzymology Volume 154, whichalso describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Oligonucleotides, e.g., for use in mutagenesis of the present invention,e.g., mutating libraries of synthetases, or altering tRNAs, aretypically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage and Caruthers,Tetrahedron Letts. 22(20):1859-1862, (1981) e.g., using an automatedsynthesizer, as described in Needham-VanDevanter et al., Nucleic AcidsRes., 12:6159-6168 (1984).

The invention also relates to eukaryotic host cells, non-eukaryotic hostcells, and organisms for the in vivo incorporation of an unnatural aminoacid via orthogonal tRNA/RS pairs. Host cells are genetically engineered(including but not limited to, transformed, transduced or transfected)with the polynucleotides of the invention or constructs which include apolynucleotide of the invention, including but not limited to, a vectorof the invention, which can be, for example, a cloning vector or anexpression vector. For example, the coding regions for the orthogonaltRNA, the orthogonal tRNA synthetase, and the protein to be derivatizedare operably linked to gene expression control elements that arefunctional in the desired host cell. The vector can be, for example, inthe form of a plasmid, a cosmid, a phage, a bacterium, a virus, a nakedpolynucleotide, or a conjugated polynucleotide. The vectors areintroduced into cells and/or microorganisms by standard methodsincluding electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA 82,5824 (1985)), infection by viral vectors, high velocity ballisticpenetration by small particles with the nucleic acid either within thematrix of small beads or particles, or on the surface (Klein et al.,Nature 327, 70-73 (1987)), and/or the like.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, screeningsteps, activating promoters or selecting transformants. These cells canoptionally be cultured into transgenic organisms. Other usefulreferences, including but not limited to for cell isolation and culture(e.g., for subsequent nucleic acid isolation) include Freshney (1994)Culture of Animal Cells, a Manual of Basic Technique, third edition,Wiley-Liss, New York and the references cited therein; Payne et al.(1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds.) (1995) PlantCell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds.)The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.

Several well-known methods of introducing target nucleic acids intocells are available, any of which can be used in the invention. Theseinclude: fusion of the recipient cells with bacterial protoplastscontaining the DNA, electroporation, projectile bombardment, andinfection with viral vectors (discussed further, below), etc. Bacterialcells can be used to amplify the number of plasmids containing DNAconstructs of this invention. The bacteria are grown to log phase andthe plasmids within the bacteria can be isolated by a variety of methodsknown in the art (see, for instance, Sambrook). In addition, kits arecommercially available for the purification of plasmids from bacteria,(see, e.g., EasyPrep™, FlexiPrep™, both from Pharmacia Biotech;StrataClean™ from Stratagene; and, QIAprep™ from Qiagen). The isolatedand purified plasmids are then further manipulated to produce otherplasmids, used to transfect cells or incorporated into related vectorsto infect organisms. Typical vectors contain transcription andtranslation terminators, transcription and translation initiationsequences, and promoters useful for regulation of the expression of theparticular target nucleic acid. The vectors optionally comprise genericexpression cassettes containing at least one independent terminatorsequence, sequences permitting replication of the cassette ineukaryotes, or prokaryotes, or both, (including but not limited to,shuttle vectors) and selection markers for both prokaryotic andeukaryotic systems. Vectors are suitable for replication and integrationin prokaryotes, eukaryotes, or both. See, Gillam & Smith, Gene 8:81(1979); Roberts, et al., Nature, 328:731 (1987); Schneider, E., et al.,Protein Expr. Purif. 6(1):10-14 (1995); Ausubel, Sambrook, Berger (allsupra). A catalogue of bacteria and bacteriophages useful for cloning isprovided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria andBacteriophage (1992) Gherna et al. (eds) published by the ATCC.Additional basic procedures for sequencing, cloning and other aspects ofmolecular biology and underlying theoretical considerations are alsofound in Watson et al. (1992) Recombinant DNA Second Edition ScientificAmerican Books, N.Y. In addition, essentially any nucleic acid (andvirtually any labeled nucleic acid, whether standard or non-standard)can be custom or standard ordered from any of a variety of commercialsources, such as the Midland Certified Reagent Company (Midland, Tex.available on the World Wide Web at mcrc.com), The Great American GeneCompany (Ramona, Calif. available on the World Wide Web at genco.com),ExpressGen Inc. (Chicago, Ill. available on the World Wide Web atexpressgen.com), Operon Technologies Inc. (Alameda, Calif.) and manyothers.

Selector Codons

Selector codons of the invention expand the genetic codon framework ofprotein biosynthetic machinery. For example, a selector codon includes,but is not limited to, a unique three base codon, a nonsense codon, suchas a stop codon, including but not limited to, an amber codon (UAG), anochre codon, or an opal codon (UGA), an unnatural codon, a four or morebase codon, a rare codon, or the like. It is readily apparent to thoseof ordinary skill in the art that there is a wide range in the number ofselector codons that can be introduced into a desired gene orpolynucleotide, including but not limited to, one or more, two or more,three or more, 4, 5, 6, 7, 8, 9, 10 or more in a single polynucleotideencoding at least a portion of the hIFN polypeptide.

In one embodiment, the methods involve the use of a selector codon thatis a stop codon for the incorporation of one or more unnatural aminoacids in vivo. For example, an O-tRNA is produced that recognizes thestop codon, including but not limited to, UAG, and is aminoacylated byan O-RS with a desired unnatural amino acid. This O-tRNA is notrecognized by the naturally occurring host's aminoacyl-tRNA synthetases.Conventional site-directed mutagenesis can be used to introduce the stopcodon, including but not limited to, TAG, at the site of interest in apolypeptide of interest. See, e.g., Sayers, J. R., et al. (1988), 5′-3′Exonucleases in phosphorothioate-based oligonucleotide-directedmutagenesis. Nucleic Acids Res, 16:791-802. When the O-RS, O-tRNA andthe nucleic acid that encodes the polypeptide of interest are combinedin vivo, the unnatural amino acid is incorporated in response to the UAGcodon to give a polypeptide containing the unnatural amino acid at thespecified position.

The incorporation of unnatural amino acids in vivo can be done withoutsignificant perturbation of the eukaryotic host cell. For example,because the suppression efficiency for the UAG codon depends upon thecompetition between the O-tRNA, including but not limited to, the ambersuppressor tRNA, and a eukaryotic release factor (including but notlimited to, eRF) (which binds to a stop codon and initiates release ofthe growing peptide from the ribosome), the suppression efficiency canbe modulated by, including but not limited to, increasing the expressionlevel of O-tRNA, and/or the suppressor tRNA.

Unnatural amino acids can also be encoded with rare codons. For example,when the arginine concentration in an in vitro protein synthesisreaction is reduced, the rare arginine codon, AGG, has proven to beefficient for insertion of Ala by a synthetic tRNA acylated withalanine. See, e.g., Ma et al., Biochemistry, 32:7939 (1993). In thiscase, the synthetic tRNA competes with the naturally occurring tRNAArg,which exists as a minor species in Escherichia coli. Some organisms donot use all triplet codons. An unassigned codon AGA in Micrococcusluteus has been utilized for insertion of amino acids in an in vitrotranscription/translation extract. See, e.g., Kowal and Oliver, Nucl.Acid. Res., 25:4685 (1997). Components of the present invention can begenerated to use these rare codons in vivo.

Selector codons also comprise extended codons, including but not limitedto, four or more base codons, such as, four, five, six or more basecodons. Examples of four base codons include, but are not limited to,AGGA, CUAG, UAGA, CCCU and the like. Examples of five base codonsinclude, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU,UAGGC and the like. A feature of the invention includes using extendedcodons based on frameshift suppression. Four or more base codons caninsert, including but not limited to, one or multiple unnatural aminoacids into the same protein. For example, in the presence of mutatedO-tRNAs, including but not limited to, a special frameshift suppressortRNAs, with anticodon loops, for example, with at least 8-10 ntanticodon loops, the four or more base codon is read as single aminoacid. In other embodiments, the anticodon loops can decode, includingbut not limited to, at least a four-base codon, at least a five-basecodon, or at least a six-base codon or more. Since there are 256possible four-base codons, multiple unnatural amino acids can be encodedin the same cell using a four or more base codon. See, Anderson et al.,(2002) Exploring the Limits of Codon and Anticodon Size, Chemistry andBiology, 9:237-244; Magliery, (2001) Expanding the Genetic Code:Selection of Efficient Suppressors of Four-base Codons andIdentification of “Shifty” Four-base Codons with a Library Approach inEscherichia coli, J. Mol. Biol. 307: 755-769.

For example, four-base codons have been used to incorporate unnaturalamino acids into proteins using in vitro biosynthetic methods. See,e.g., Ma et al., (1993) Biochemistry, 32:7939; and Hohsaka et al.,(1999) J. Am. Chem. Soc., 121:34. CGGG and AGGU were used tosimultaneously incorporate 2-naphthylalanine and an NBD derivative oflysine into streptavidin in vitro with two chemically acylatedframeshift suppressor tRNAs. See, e.g., Hohsaka et al., (1999) J. Am.Chem. Soc., 121:12194. In an in vivo study, Moore et al. examined theability of tRNALeu derivatives with NCUA anticodons to suppress UAGNcodons (N can be U, A, G, or C), and found that the quadruplet UAGA canbe decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13to 26% with little decoding in the 0 or −1 frame. See, Moore et al.,(2000) J. Mol. Biol. 298:195. In one embodiment, extended codons basedon rare codons or nonsense codons can be used in the present invention,which can reduce missense readthrough and frameshift suppression atother unwanted sites.

For a given system, a selector codon can also include one of the naturalthree base codons, where the endogenous system does not use (or rarelyuses) the natural base codon. For example, this includes a system thatis lacking a tRNA that recognizes the natural three base codon, and/or asystem where the three base codon is a rare codon.

Selector codons optionally include unnatural base pairs. These unnaturalbase pairs further expand the existing genetic alphabet. One extra basepair increases the number of triplet codons from 64 to 125. Propertiesof third base pairs include stable and selective base pairing, efficientenzymatic incorporation into DNA with high fidelity by a polymerase, andthe efficient continued primer extension after synthesis of the nascentunnatural base pair. Descriptions of unnatural base pairs which can beadapted for methods and compositions include, e.g., Hirao, et al.,(2002) An unnatural base pair for incorporating amino acid analoguesinto protein, Nature Biotechnology, 20:177-182. See, also, Wu, Y., etal., (2002) J. Am. Chem. Soc. 124:14626-14630. Other relevantpublications are listed below.

For in vivo usage, the unnatural nucleoside is membrane permeable and isphosphorylated to form the corresponding triphosphate. In addition, theincreased genetic information is stable and not destroyed by cellularenzymes. Previous efforts by Benner and others took advantage ofhydrogen bonding patterns that are different from those in canonicalWatson-Crick pairs, the most noteworthy example of which is theiso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc.,111:8322; and Piccirilli et al., (1990) Nature, 343:33; Kool, (2000)Curr. Opin. Chem. Biol., 4:602. These bases in general mispair to somedegree with natural bases and cannot be enzymatically replicated. Kooland co-workers demonstrated that hydrophobic packing interactionsbetween bases can replace hydrogen bonding to drive the formation ofbase pair. See, Kool, (2000) Curr. Opin. Chem. Biol., 4:602; and Guckianand Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In an effort todevelop an unnatural base pair satisfying all the above requirements,Schultz, Romesberg and co-workers have systematically synthesized andstudied a series of unnatural hydrophobic bases. A PICS:PICS self-pairis found to be more stable than natural base pairs, and can beefficiently incorporated into DNA by Klenow fragment of Escherichia coliDNA polymerase I (KF). See, e.g., McMinn et al., (1999) J. Am. Chem.Soc., 121:11585-6; and Ogawa et al., (2000) J. Am. Chem. Soc., 122:3274.A 3MN:3MN self-pair can be synthesized by KF with efficiency andselectivity sufficient for biological function. See, e.g., Ogawa et al.,(2000) J. Am. Chem. Soc., 122:8803. However, both bases act as a chainterminator for further replication. A mutant DNA polymerase has beenrecently evolved that can be used to replicate the PICS self pair. Inaddition, a 7AI self pair can be replicated. See, e.g., Tae et al.,(2001) J. Am. Chem. Soc., 123:7439. A novel metallobase pair, Dipic:Py,has also been developed, which forms a stable pair upon binding Cu(II).See, Meggers et al., (2000) J. Am. Chem. Soc., 122:10714. Becauseextended codons and unnatural codons are intrinsically orthogonal tonatural codons, the methods of the invention can take advantage of thisproperty to generate orthogonal tRNAs for them.

A translational bypassing system can also be used to incorporate anunnatural amino acid in a desired polypeptide. In a translationalbypassing system, a large sequence is incorporated into a gene but isnot translated into protein. The sequence contains a structure thatserves as a cue to induce the ribosome to hop over the sequence andresume translation downstream of the insertion.

In certain embodiments, the protein or polypeptide of interest (orportion thereof) in the methods and/or compositions of the invention isencoded by a nucleic acid. Typically, the nucleic acid comprises atleast one selector codon, at least two selector codons, at least threeselector codons, at least four selector codons, at least five selectorcodons, at least six selector codons, at least seven selector codons, atleast eight selector codons, at least nine selector codons, ten or moreselector codons.

Genes coding for proteins or polypeptides of interest can be mutagenizedusing methods well-known to one of ordinary skill in the art anddescribed herein to include, for example, one or more selector codon forthe incorporation of an unnatural amino acid. For example, a nucleicacid for a protein of interest is mutagenized to include one or moreselector codon, providing for the incorporation of one or more unnaturalamino acids. The invention includes any such variant, including but notlimited to, mutant, versions of any protein, for example, including atleast one unnatural amino acid. Similarly, the invention also includescorresponding nucleic acids, i.e., any nucleic acid with one or moreselector codon that encodes one or more unnatural amino acid.

Nucleic acid molecules encoding a protein of interest such as a hIFNpolypeptide may be readily mutated to introduce a cysteine at anydesired position of the polypeptide. Cysteine is widely used tointroduce reactive molecules, water soluble polymers, proteins, or awide variety of other molecules, onto a protein of interest. Methodssuitable for the incorporation of cysteine into a desired position of apolypeptide are known to those of ordinary skill in the art, such asthose described in U.S. Pat. No. 6,608,183, which is incorporated byreference herein, and standard mutagenesis techniques.

IV. Non-Naturally Encoded Amino Acids

A very wide variety of non-naturally encoded amino acids are suitablefor use in the present invention. Any number of non-naturally encodedamino acids can be introduced into a hIFN polypeptide. In general, theintroduced non-naturally encoded amino acids are substantiallychemically inert toward the 20 common, genetically-encoded amino acids(i.e., alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine). In some embodiments, thenon-naturally encoded amino acids include side chain functional groupsthat react efficiently and selectively with functional groups not foundin the 20 common amino acids (including but not limited to, azido,ketone, aldehyde and aminooxy groups) to form stable conjugates. Forexample, a hIFN polypeptide that includes a non-naturally encoded aminoacid containing an azido functional group can be reacted with a polymer(including but not limited to, poly(ethylene glycol) or, alternatively,a second polypeptide containing an alkyne moiety to form a stableconjugate resulting for the selective reaction of the azide and thealkyne functional groups to form a Huisgen [3+2]cycloaddition product.

The generic structure of an alpha-amino acid is illustrated as follows(Formula I):

A non-naturally encoded amino acid is typically any structure having theabove-listed formula wherein the R group is any substituent other thanone used in the twenty natural amino acids, and may be suitable for usein the present invention. Because the non-naturally encoded amino acidsof the invention typically differ from the natural amino acids only inthe structure of the side chain, the non-naturally encoded amino acidsform amide bonds with other amino acids, including but not limited to,natural or non-naturally encoded, in the same manner in which they areformed in naturally occurring polypeptides. However, the non-naturallyencoded amino acids have side chain groups that distinguish them fromthe natural amino acids. For example, R optionally comprises an alkyl-,aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-,hydrazide, alkenyl, alkynl, ether, thiol, seleno-, sulfonyl-, borate,boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine,aldehyde, ester, thioacid, hydroxylamine, amino group, or the like orany combination thereof. Other non-naturally occurring amino acids ofinterest that may be suitable for use in the present invention include,but are not limited to, amino acids comprising a photoactivatablecross-linker, spin-labeled amino acids, fluorescent amino acids, metalbinding amino acids, metal-containing amino acids, radioactive aminoacids, amino acids with novel functional groups, amino acids thatcovalently or noncovalently interact with other molecules, photocagedand/or photoisomerizable amino acids, amino acids comprising biotin or abiotin analogue, glycosylated amino acids such as a sugar substitutedserine, other carbohydrate modified amino acids, keto-containing aminoacids, amino acids comprising polyethylene glycol or polyether, heavyatom substituted amino acids, chemically cleavable and/or photocleavableamino acids, amino acids with an elongated side chains as compared tonatural amino acids, including but not limited to, polyethers or longchain hydrocarbons, including but not limited to, greater than about 5or greater than about 10 carbons, carbon-linked sugar-containing aminoacids, redox-active amino acids, amino thioacid containing amino acids,and amino acids comprising one or more toxic moiety.

Exemplary non-naturally encoded amino acids that may be suitable for usein the present invention and that are useful for reactions with watersoluble polymers include, but are not limited to, those with carbonyl,aminooxy, hydrazine, hydrazide, semicarbazide, azide and alkyne reactivegroups. In some embodiments, non-naturally encoded amino acids comprisea saccharide moiety. Examples of such amino acids includeN-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine,N-acetyl-L-glucosaminyl-L-threonine,N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.Examples of such amino acids also include examples where thenaturally-occurring N— or O— linkage between the amino acid and thesaccharide is replaced by a covalent linkage not commonly found innature—including but not limited to, an alkene, an oxime, a thioether,an amide and the like. Examples of such amino acids also includesaccharides that are not commonly found in naturally-occurring proteinssuch as 2-deoxy-glucose, 2-deoxygalactose and the like.

Many of the non-naturally encoded amino acids provided herein arecommercially available, e.g., from Sigma-Aldrich (St. Louis, Mo., USA),Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), orPeptech (Burlington, Mass., USA). Those that are not commerciallyavailable are optionally synthesized as provided herein or usingstandard methods known to those of ordinary skill in the art. Fororganic synthesis techniques, see, e.g., Organic Chemistry by Fessendonand Fessendon, (1982, Second Edition, Willard Grant Press, BostonMass.); Advanced Organic Chemistry by March (Third Edition, 1985, Wileyand Sons, New York); and Advanced Organic Chemistry by Carey andSundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York).See, also, U.S. Pat. No. 7,045,337 and U.S. Patent ApplicationPublication 2003/0108885, which are incorporated by reference herein. Inaddition to unnatural amino acids that contain novel side chains,unnatural amino acids that may be suitable for use in the presentinvention also optionally comprise modified backbone structures,including but not limited to, as illustrated by the structures ofFormula II and III:

wherein Z typically comprises OH, NH₂, SH, NH—R′, or S—R′; X and Y,which can be the same or different, typically comprise S or O, and R andR′, which are optionally the same or different, are typically selectedfrom the same list of constituents for the R group described above forthe unnatural amino acids having Formula I as well as hydrogen. Forexample, unnatural amino acids of the invention optionally comprisesubstitutions in the amino or carboxyl group as illustrated by FormulasII and III. Unnatural amino acids of this type include, but are notlimited to, α-hydroxy acids, α-thioacids, α-aminothiocarboxylates,including but not limited to, with side chains corresponding to thecommon twenty natural amino acids or unnatural side chains. In addition,substitutions at the α-carbon optionally include, but are not limitedto, L, D, or α-α-disubstituted amino acids such as D-glutamate,D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like. Otherstructural alternatives include cyclic amino acids, such as prolineanalogues as well as 3, 4, 6, 7, 8, and 9 membered ring prolineanalogues, β and γ amino acids such as substituted β-alanine and γ-aminobutyric acid.

Many unnatural amino acids are based on natural amino acids, such astyrosine, glutamine, phenylalanine, and the like, and are suitable foruse in the present invention. Tyrosine analogs include, but are notlimited to, para-substituted tyrosines, ortho-substituted tyrosines, andmeta substituted tyrosines, where the substituted tyrosine comprises,including but not limited to, a keto group (including but not limitedto, an acetyl group), a benzoyl group, an amino group, a hydrazine, anhydroxyamine, a thiol group, a carboxy group, an isopropyl group, amethyl group, a C₆-C₂₀ straight chain or branched hydrocarbon, asaturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a nitro group, an alkynyl group or the like. In addition,multiply substituted aryl rings are also contemplated. Glutamine analogsthat may be suitable for use in the present invention include, but arenot limited to, α-hydroxy derivatives, γ-substituted derivatives, cyclicderivatives, and amide substituted glutamine derivatives. Examplephenylalanine analogs that may be suitable for use in the presentinvention include, but are not limited to, para-substitutedphenylalanines, ortho-substituted phenyalanines, and meta-substitutedphenylalanines, where the substituent comprises, including but notlimited to, a hydroxy group, a methoxy group, a methyl group, an allylgroup, an aldehyde, an azido, an iodo, a bromo, a keto group (includingbut not limited to, an acetyl group), a benzoyl, an alkynyl group, orthe like. Specific examples of unnatural amino acids that may besuitable for use in the present invention include, but are not limitedto, a p-acetyl-L-phenylalanine, an O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, anisopropyl-L-phenylalanine, and a p-propargyloxy-phenylalanine, and thelike. Examples of structures of a variety of unnatural amino acids thatmay be suitable for use in the present invention are provided in, forexample, WO 2002/085923 entitled “In vivo incorporation of unnaturalamino acids.” See also Kiick et al., (2002) Incorporation of azides intorecombinant proteins for chemoselective modification by the Staudingerligation, PNAS 99:19-24, which is incorporated by reference herein, foradditional methionine analogs.

In one embodiment, compositions of a hIFN polypeptide that include anunnatural amino acid (such as p-(propargyloxy)-phenyalanine) areprovided. Various compositions comprising p-(propargyloxy)-phenyalanineand, including but not limited to, proteins and/or cells, are alsoprovided. In one aspect, a composition that includes thep-(propargyloxy)-phenyalanine unnatural amino acid, further includes anorthogonal tRNA. The unnatural amino acid can be bonded (including butnot limited to, covalently) to the orthogonal tRNA, including but notlimited to, covalently bonded to the orthogonal tRNA though anamino-acyl bond, covalently bonded to a 3′OH or a 2′OH of a terminalribose sugar of the orthogonal tRNA, etc.

The chemical moieties via unnatural amino acids that can be incorporatedinto proteins offer a variety of advantages and manipulations of theprotein. For example, the unique reactivity of a keto functional groupallows selective modification of proteins with any of a number ofhydrazine- or hydroxylamine-containing reagents in vitro and in vivo. Aheavy atom unnatural amino acid, for example, can be useful for phasingX-ray structure data. The site-specific introduction of heavy atomsusing unnatural amino acids also provides selectivity and flexibility inchoosing positions for heavy atoms. Photoreactive unnatural amino acids(including but not limited to, amino acids with benzophenone andarylazides (including but not limited to, phenylazide) side chains), forexample, allow for efficient in vivo and in vitro photocrosslinking ofprotein. Examples of photoreactive unnatural amino acids include, butare not limited to, p-azido-phenylalanine and p-benzoyl-phenylalanine.The protein with the photoreactive unnatural amino acids can then becrosslinked at will by excitation of the photoreactive group-providingtemporal control. In one example, the methyl group of an unnatural aminocan be substituted with an isotopically labeled, including but notlimited to, methyl group, as a probe of local structure and dynamics,including but not limited to, with the use of nuclear magnetic resonanceand vibrational spectroscopy. Alkynyl or azido functional groups, forexample, allow the selective modification of proteins with moleculesthrough a [3+2]cycloaddition reaction.

A non-natural amino acid incorporated into a polypeptide at the aminoterminus can be composed of an R group that is any substituent otherthan one used in the twenty natural amino acids and a 2^(nd) reactivegroup different from the NH₂ group normally present in α-amino acids(see Formula I). A similar non-natural amino acid can be incorporated atthe carboxyl terminus with a 2^(nd) reactive group different from theCOOH group normally present in α-amino acids (see Formula I).

The unnatural amino acids of the invention may be selected or designedto provide additional characteristics unavailable in the twenty naturalamino acids. For example, unnatural amino acid may be optionallydesigned or selected to modify the biological properties of a protein,e.g., into which they are incorporated. For example, the followingproperties may be optionally modified by inclusion of an unnatural aminoacid into a protein: toxicity, biodistribution, solubility, stability,e.g., thermal, hydrolytic, oxidative, resistance to enzymaticdegradation, and the like, facility of purification and processing,structural properties, spectroscopic properties, chemical and/orphotochemical properties, catalytic activity, redox potential,half-life, ability to react with other molecules, e.g., covalently ornoncovalently, and the like.

Chemical Synthesis of Unnatural Amino Acids

Many of the unnatural amino acids suitable for use in the presentinvention are commercially available, e.g., from Sigma (USA) or Aldrich(Milwaukee, Wis., USA). Those that are not commercially available areoptionally synthesized as provided herein or as provided in variouspublications or using standard methods known to those of ordinary skillin the art. For organic synthesis techniques, see, e.g., OrganicChemistry by Fessendon and Fessendon, (1982, Second Edition, WillardGrant Press, Boston Mass.); Advanced Organic Chemistry by March (ThirdEdition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistryby Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press,New York). Additional publications describing the synthesis of unnaturalamino acids include, e.g., WO 2002/085923 entitled “In vivoincorporation of Unnatural Amino Acids;” Matsoukas et al., (1995) J.Med. Chem., 38, 4660-4669; King, F. E. & Kidd, D. A. A. (1949) A NewSynthesis of Glutamine and of γ-Dipeptides of Glutamic Acid fromPhthylated Intermediates. J. Chem. Soc. 3315-3319; Friedman, O. M. &Chatterrji, R. (1959) Synthesis of Derivatives of Glutamine as ModelSubstrates for Anti-Tumor Agents. J. Am. Chem. Soc. 81, 3750-3752;Craig, J. C. et al. (1988) Absolute Configuration of the Enantiomers of7-Chloro-4 [[4-(diethylamino)-1-methylbutyl]amino]quinoline(Chloroquine). J. Org. Chem. 53, 1167-1170; Azoulay, M., Vilmont, M. &Frappier, F. (1991) Glutamine analogues as Potential Antimalarials, Eur.J. Med. Chem. 26, 201-5; Koskinen, A. M. P. & Rapoport, H. (1989)Synthesis of 4-Substituted Prolines as Conformationally ConstrainedAmino Acid Analogues. J. Org. Chem. 54, 1859-1866; Christie, B. D. &Rapoport, H. (1985) Synthesis of Optically Pure Pipecolates fromL-Asparagine. Application to the Total Synthesis of (+)-Apovincaminethrough Amino Acid Decarbonylation and Iminium Ion Cyclization. J. Org.Chem. 50:1239-1246; Barton et al., (1987) Synthesis of Novelalpha-Amino-Acids and Derivatives Using Radical Chemistry. Synthesis ofL- and D-alpha-Amino-Adipic Acids, L-alpha-aminopimelic Acid andAppropriate Unsaturated Derivatives. Tetrahedron 43:4297-4308; and,Subasinghe et al., (1992) Quisqualic acid analogues: synthesis ofbeta-heterocyclic 2-aminopropanoic acid derivatives and their activityat a novel quisqualate-sensitized site. J. Med. Chem. 35:4602-7. Seealso, U.S. Patent Publication No. US 2004/0198637 entitled “ProteinArrays,” which is incorporated by reference herein.

A. Carbonyl Reactive Groups

Amino acids with a carbonyl reactive group allow for a variety ofreactions to link molecules (including but not limited to, PEG or otherwater soluble molecules) via nucleophilic addition or aldol condensationreactions among others.

Exemplary carbonyl-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R₂ is H, alkyl, aryl, substituted alkyl, andsubstituted aryl; and R₃ is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R₄ is H, an amino acid, a polypeptide,or a carboxy terminus modification group. In some embodiments, n is 1,R₁ is phenyl and R₂ is a simple alkyl (i.e., methyl, ethyl, or propyl)and the ketone moiety is positioned in the para position relative to thealkyl side chain. In some embodiments, n is 1, R₁ is phenyl and R₂ is asimple alkyl (i.e., methyl, ethyl, or propyl) and the ketone moiety ispositioned in the meta position relative to the alkyl side chain.

The synthesis of p-acetyl-(+/−)-phenylalanine andm-acetyl-(+/−)-phenylalanine is described in Zhang, Z., et al.,Biochemistry 42: 6735-6746 (2003), which is incorporated by referenceherein. Other carbonyl-containing amino acids can be similarly preparedby one of ordinary skill in the art.

In some embodiments, a polypeptide comprising a non-naturally encodedamino acid is chemically modified to generate a reactive carbonylfunctional group. For instance, an aldehyde functionality useful forconjugation reactions can be generated from a functionality havingadjacent amino and hydroxyl groups. Where the biologically activemolecule is a polypeptide, for example, an N-terminal serine orthreonine (which may be normally present or may be exposed via chemicalor enzymatic digestion) can be used to generate an aldehydefunctionality under mild oxidative cleavage conditions using periodate.See, e.g., Gaertner, et al., Bioconjug. Chem. 3: 262-268 (1992);Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992); Gaertneret al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known inthe art are restricted to the amino acid at the N-terminus of thepeptide or protein.

In the present invention, a non-naturally encoded amino acid bearingadjacent hydroxyl and amino groups can be incorporated into thepolypeptide as a “masked” aldehyde functionality. For example,5-hydroxylysine bears a hydroxyl group adjacent to the epsilon amine.Reaction conditions for generating the aldehyde typically involveaddition of molar excess of sodium metaperiodate under mild conditionsto avoid oxidation at other sites within the polypeptide. The pH of theoxidation reaction is typically about 7.0. A typical reaction involvesthe addition of about 1.5 molar excess of sodium meta periodate to abuffered solution of the polypeptide, followed by incubation for about10 minutes in the dark. See, e.g. U.S. Pat. No. 6,423,685, which isincorporated by reference herein.

The carbonyl functionality can be reacted selectively with a hydrazine-,hydrazide-, hydroxylamine-, or semicarbazide-containing reagent undermild conditions in aqueous solution to form the corresponding hydrazone,oxime, or semicarbazone linkages, respectively, that are stable underphysiological conditions. See, e.g., Jencks, W. P., J. Am. Chem. Soc.81, 475-481 (1959); Shao, J. and Tam, J. P., J. Am. Chem. Soc.117:3893-3899 (1995). Moreover, the unique reactivity of the carbonylgroup allows for selective modification in the presence of the otheramino acid side chains. See, e.g., Cornish, V. W., et al., J. Am. Chem.Soc. 118:8150-8151 (1996); Geoghegan, K. F. & Stroh, J. G., Bioconjug.Chem. 3:138-146 (1992); Mahal, L. K., et al., Science 276:1125-1128(1997).

B. Hydrazine, Hydrazide or Semicarbazide Reactive Groups

Non-naturally encoded amino acids containing a nucleophilic group, suchas a hydrazine, hydrazide or semicarbazide, allow for reaction with avariety of electrophilic groups to form conjugates (including but notlimited to, with PEG or other water soluble polymers).

Exemplary hydrazine, hydrazide or semicarbazide-containing amino acidscan be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X, is O, N, or S or not present; R₂ isH, an amino acid, a polypeptide, or an amino terminus modificationgroup, and R₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, n is 4, R₁ is not present, and X is N. In someembodiments, n is 2, R₁ is not present, and X is not present. In someembodiments, n is 1, R₁ is phenyl, X is O, and the oxygen atom ispositioned para to the alphatic group on the aryl ring.

Hydrazide-, hydrazine-, and semicarbazide-containing amino acids areavailable from commercial sources. For instance, L-glutamate-γ-hydrazideis available from Sigma Chemical (St. Louis, Mo.). Other amino acids notavailable commercially can be prepared by one of ordinary skill in theart. See, e.g., U.S. Pat. No. 6,281,211, which is incorporated byreference herein.

Polypeptides containing non-naturally encoded amino acids that bearhydrazide, hydrazine or semicarbazide functionalities can be reactedefficiently and selectively with a variety of molecules that containaldehydes or other functional groups with similar chemical reactivity.See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995).The unique reactivity of hydrazide, hydrazine and semicarbazidefunctional groups makes them significantly more reactive towardaldehydes, ketones and other electrophilic groups as compared to thenucleophilic groups present on the 20 common amino acids (including butnot limited to, the hydroxyl group of serine or threonine or the aminogroups of lysine and the N-terminus).

C. Aminooxy-Containing Amino Acids

Non-naturally encoded amino acids containing an aminooxy (also called ahydroxylamine) group allow for reaction with a variety of electrophilicgroups to form conjugates (including but not limited to, with PEG orother water soluble polymers). Like hydrazines, hydrazides andsemicarbazides, the enhanced nucleophilicity of the aminooxy grouppermits it to react efficiently and selectively with a variety ofmolecules that contain aldehydes or other functional groups with similarchemical reactivity. See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc.117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34:727-736 (2001). Whereas the result of reaction with a hydrazine group isthe corresponding hydrazone, however, an oxime results generally fromthe reaction of an aminooxy group with a carbonyl-containing group suchas a ketone.

Exemplary amino acids containing aminooxy groups can be represented asfollows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X is O, N, S or not present; m is 0-10;Y═C(O) or not present; R₂ is H, an amino acid, a polypeptide, or anamino terminus modification group, and R₃ is H, an amino acid, apolypeptide, or a carboxy terminus modification group. In someembodiments, n is 1, R₁ is phenyl, X is O, m is 1, and Y is present. Insome embodiments, n is 2, R₁ and X are not present, m is 0, and Y is notpresent.

Aminooxy-containing amino acids can be prepared from readily availableamino acid precursors (homoserine, serine and threonine). See, e.g., M.Carrasco and R. Brown, J. Org. Chem. 68: 8853-8858 (2003). Certainaminooxy-containing amino acids, such as L-2-amino-4-(aminooxy)butyricacid), have been isolated from natural sources (Rosenthal, G., Life Sci.60: 1635-1641 (1997). Other aminooxy-containing amino acids can beprepared by one of ordinary skill in the art.

D. Azide and Alkyne Reactive Groups

The unique reactivity of azide and alkyne functional groups makes themextremely useful for the selective modification of polypeptides andother biological molecules. Organic azides, particularly alphaticazides, and alkynes are generally stable toward common reactive chemicalconditions. In particular, both the azide and the alkyne functionalgroups are inert toward the side chains (i.e., R groups) of the 20common amino acids found in naturally-occurring polypeptides. Whenbrought into close proximity, however, the “spring-loaded” nature of theazide and alkyne groups is revealed and they react selectively andefficiently via Huisgen [3+2] cycloaddition reaction to generate thecorresponding triazole. See, e.g., Chin J., et al., Science 301:964-7(2003); Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Chin,J. W., et al., J. Am. Chem. Soc. 124:9026-9027 (2002).

Because the Huisgen cycloaddition reaction involves a selectivecycloaddition reaction (see, e.g., Padwa, A., in COMPREHENSIVE ORGANICSYNTHESIS, Vol. 4, (ed. Trost, B. M., 1991), p. 1069-1109; Huisgen, R.in 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A., 1984), p. 1-176)rather than a nucleophilic substitution, the incorporation ofnon-naturally encoded amino acids bearing azide and alkyne-containingside chains permits the resultant polypeptides to be modifiedselectively at the position of the non-naturally encoded amino acid.Cycloaddition reaction involving azide or alkyne-containing hIFNpolypeptide can be carried out at room temperature under aqueousconditions by the addition of Cu(II) (including but not limited to, inthe form of a catalytic amount of CuSO₄) in the presence of a reducingagent for reducing Cu(II) to Cu(I), in situ, in catalytic amount. See,e.g., Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Tornoe,C. W., et al., J. Org. Chem. 67:3057-3064 (2002); Rostovtsev, et al.,Angew. Chem. Int. Ed. 41:2596-2599 (2002). Exemplary reducing agentsinclude, including but not limited to, ascorbate, metallic copper,quinine, hydroquinone, vitamin K, glutathione, cysteine, Fe²⁺, Co²⁺, andan applied electric potential.

In some cases, where a Huisgen [3+2]cycloaddition reaction between anazide and an alkyne is desired, the hIFN polypeptide comprises anon-naturally encoded amino acid comprising an alkyne moiety and thewater soluble polymer to be attached to the amino acid comprises anazide moiety. Alternatively, the converse reaction (i.e., with the azidemoiety on the amino acid and the alkyne moiety present on the watersoluble polymer) can also be performed.

The azide functional group can also be reacted selectively with a watersoluble polymer containing an aryl ester and appropriatelyfunctionalized with an aryl phosphine moiety to generate an amidelinkage. The aryl phosphine group reduces the azide in situ and theresulting amine then reacts efficiently with a proximal ester linkage togenerate the corresponding amide. See, e.g., E. Saxon and C. Bertozzi,Science 287, 2007-2010 (2000). The azide-containing amino acid can beeither an alkyl azide (including but not limited to,2-amino-6-azido-1-hexanoic acid) or an aryl azide(p-azido-phenylalanine).

Exemplary water soluble polymers containing an aryl ester and aphosphine moiety can be represented as follows:

wherein X can be O, N, S or not present, Ph is phenyl, W is a watersoluble polymer and R can be H, alkyl, aryl, substituted alkyl andsubstituted aryl groups. Exemplary R groups include but are not limitedto —CH₂, —C(CH₃)₃, —OR′, —NR′R″, —SR, -halogen, —C(O)R′, —CONR′R″,—S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂. R′, R″, R′″ and R′″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R′″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

The azide functional group can also be reacted selectively with a watersoluble polymer containing a thioester and appropriately functionalizedwith an aryl phosphine moiety to generate an amide linkage. The arylphosphine group reduces the azide in situ and the resulting amine thenreacts efficiently with the thioester linkage to generate thecorresponding amide. Exemplary water soluble polymers containing athioester and a phosphine moiety can be represented as follows:

wherein n is 1-10; X can be O, N, S or not present, Ph is phenyl, and Wis a water soluble polymer.

Exemplary alkyne-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X is O, N, S or not present; m is 0-10,R₂ is H, an amino acid, a polypeptide, or an amino terminus modificationgroup, and R₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group. In some embodiments, n is 1, R₁ is phenyl, X is notpresent, m is 0 and the acetylene moiety is positioned in the paraposition relative to the alkyl side chain. In some embodiments, n is 1,R₁ is phenyl, X is O, m is 1 and the propargyloxy group is positioned inthe para position relative to the alkyl side chain (i.e.,O-propargyl-tyrosine). In some embodiments, n is 1, R₁ and X are notpresent and m is 0 (i.e., proparylglycine).

Alkyne-containing amino acids are commercially available. For example,propargylglycine is commercially available from Peptech (Burlington,Mass.). Alternatively, alkyne-containing amino acids can be preparedaccording to standard methods. For instance, p-propargyloxyphenylalaninecan be synthesized, for example, as described in Deiters, A., et al., J.Am. Chem. Soc. 125: 11782-11783 (2003), and 4-alkynyl-L-phenylalaninecan be synthesized as described in Kayser, B., et al., Tetrahedron53(7): 2475-2484 (1997). Other alkyne-containing amino acids can beprepared by one of ordinary skill in the art.

Exemplary azide-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; m is 0-10; R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group. In some embodiments, n is 1, R₁ is phenyl, X is notpresent, m is 0 and the azide moiety is positioned para to the alkylside chain. In some embodiments, n is 0-4 and R₁ and X are not present,and m=0. In some embodiments, n is 1, R₁ is phenyl, X is O, m is 2 andthe β-azidoethoxy moiety is positioned in the para position relative tothe alkyl side chain.

Azide-containing amino acids are available from commercial sources. Forinstance, 4-azidophenylalanine can be obtained from Chem-ImpexInternational, Inc. (Wood Dale, Ill.). For those azide-containing aminoacids that are not commercially available, the azide group can beprepared relatively readily using standard methods known to those ofordinary skill in the art, including but not limited to, viadisplacement of a suitable leaving group (including but not limited to,halide, mesylate, tosylate) or via opening of a suitably protectedlactone. See, e.g., Advanced Organic Chemistry by March (Third Edition,1985, Wiley and Sons, New York).

E. Aminothiol Reactive Groups

The unique reactivity of beta-substituted aminothiol functional groupsmakes them extremely useful for the selective modification ofpolypeptides and other biological molecules that contain aldehyde groupsvia formation of the thiazolidine. See, e.g., J. Shao and J. Tam, J. Am.Chem. Soc. 1995, 117 (14) 3893-3899. In some embodiments,beta-substituted aminothiol amino acids can be incorporated into hIFNpolypeptides and then reacted with water soluble polymers comprising analdehyde functionality. In some embodiments, a water soluble polymer,drug conjugate or other payload can be coupled to a hIFN polypeptidecomprising a beta-substituted aminothiol amino acid via formation of thethiazolidine.

Cellular Uptake of Unnatural Amino Acids

Unnatural amino acid uptake by a cell is one issue that is typicallyconsidered when designing and selecting unnatural amino acids, includingbut not limited to, for incorporation into a protein. For example, thehigh charge density of (X-amino acids suggests that these compounds areunlikely to be cell permeable. Natural amino acids are taken up into theeukaryotic cell via a collection of protein-based transport systems. Arapid screen can be done which assesses which unnatural amino acids, ifany, are taken up by cells. See, e.g., the toxicity assays in, e.g.,U.S. Patent Publication No. US 2004/0198637 entitled “Protein Arrays”which is incorporated by reference herein; and Liu, D. R. & Schultz, P.G. (1999) Progress toward the evolution of an organism with an expandedgenetic code. PNAS United States 96:4780-4785. Although uptake is easilyanalyzed with various assays, an alternative to designing unnaturalamino acids that are amenable to cellular uptake pathways is to providebiosynthetic pathways to create amino acids in vivo.

Biosynthesis of Unnatural Amino Acids

Many biosynthetic pathways already exist in cells for the production ofamino acids and other compounds. While a biosynthetic method for aparticular unnatural amino acid may not exist in nature, including butnot limited to, in a cell, the invention provides such methods. Forexample, biosynthetic pathways for unnatural amino acids are optionallygenerated in host cell by adding new enzymes or modifying existing hostcell pathways. Additional new enzymes are optionally naturally occurringenzymes or artificially evolved enzymes. For example, the biosynthesisof p-aminophenylalanine (as presented in an example in WO 2002/085923entitled “In vivo incorporation of unnatural amino acids”) relies on theaddition of a combination of known enzymes from other organisms. Thegenes for these enzymes can be introduced into a eukaryotic cell bytransforming the cell with a plasmid comprising the genes. The genes,when expressed in the cell, provide an enzymatic pathway to synthesizethe desired compound. Examples of the types of enzymes that areoptionally added are provided in the examples below. Additional enzymessequences are found, for example, in Genbank. Artificially evolvedenzymes are also optionally added into a cell in the same manner. Inthis manner, the cellular machinery and resources of a cell aremanipulated to produce unnatural amino acids.

A variety of methods are available for producing novel enzymes for usein biosynthetic pathways or for evolution of existing pathways. Forexample, recursive recombination, including but not limited to, asdeveloped by Maxygen, Inc. (available on the World Wide Web atmaxygen.com), is optionally used to develop novel enzymes and pathways.See, e.g., Stemmer (1994), Rapid evolution of a protein in vitro by DNAshuffling, Nature 370(4):389-391; and, Stemmer, (1994), DNA shuffling byrandom fragmentation and reassembly. In vitro recombination formolecular evolution, Proc. Natl. Acad. Sci. USA., 91:10747-10751.Similarly DesignPath™, developed by Genencor (available on the WorldWide Web at genencor.com) is optionally used for metabolic pathwayengineering, including but not limited to, to engineer a pathway tocreate O-methyl-L-tyrosine in a cell. This technology reconstructsexisting pathways in host organisms using a combination of new genes,including but not limited to, those identified through functionalgenomics, and molecular evolution and design. Diversa Corporation(available on the World Wide Web at diversa.com) also providestechnology for rapidly screening libraries of genes and gene pathways,including but not limited to, to create new pathways.

Typically, the unnatural amino acid produced with an engineeredbiosynthetic pathway of the invention is produced in a concentrationsufficient for efficient protein biosynthesis, including but not limitedto, a natural cellular amount, but not to such a degree as to affect theconcentration of the other amino acids or exhaust cellular resources.Typical concentrations produced in vivo in this manner are about 10 mMto about 0.05 mM. Once a cell is transformed with a plasmid comprisingthe genes used to produce enzymes desired for a specific pathway and anunnatural amino acid is generated, in vivo selections are optionallyused to further optimize the production of the unnatural amino acid forboth ribosomal protein synthesis and cell growth.

Polypeptides with Unnatural Amino Acids

The incorporation of an unnatural amino acid can be done for a varietyof purposes, including but not limited to, tailoring changes in proteinstructure and/or function, changing size, acidity, nucleophilicity,hydrogen bonding, hydrophobicity, accessibility of protease targetsites, targeting to a moiety (including but not limited to, for aprotein array), adding a biologically active molecule, attaching apolymer, attaching a radionuclide, modulating serum half-life,modulating tissue penetration (e.g. tumors), modulating activetransport, modulating tissue, cell or organ specificity or distribution(e.g. liver), modulating immunogenicity, modulating protease resistance,etc. Alterations in signal transduction may be achieved through sitespecific PEGylation of hIFN. Proteins that include an unnatural aminoacid can have enhanced or even entirely new catalytic or biophysicalproperties. For example, the following properties are optionallymodified by inclusion of an unnatural amino acid into a protein:toxicity, biodistribution, structural properties, spectroscopicproperties, chemical and/or photochemical properties, catalytic ability,half-life (including but not limited to, serum half-life), ability toreact with other molecules, including but not limited to, covalently ornoncovalently, and the like. The compositions including proteins thatinclude at least one unnatural amino acid are useful for, including butnot limited to, novel therapeutics, diagnostics, catalytic enzymes,industrial enzymes, binding proteins (including but not limited to,antibodies), and including but not limited to, the study of proteinstructure and function. See, e.g., Dougherty, (2000) Unnatural AminoAcids as Probes of Protein Structure and Function, Current Opinion inChemical Biology, 4:645-652.

In one aspect of the invention, a composition includes at least oneprotein with at least one, including but not limited to, at least two,at least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, or at least ten or more unnaturalamino acids. The unnatural amino acids can be the same or different,including but not limited to, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 or more different sites in the protein that comprise 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 or more different unnatural amino acids. In anotheraspect, a composition includes a protein with at least one, but fewerthan all, of a particular amino acid present in the protein issubstituted with the unnatural amino acid. For a given protein with morethan one unnatural amino acids, the unnatural amino acids can beidentical or different (including but not limited to, the protein caninclude two or more different types of unnatural amino acids, or caninclude two of the same unnatural amino acid). For a given protein withmore than two unnatural amino acids, the unnatural amino acids can bethe same, different or a combination of a multiple unnatural amino acidof the same kind with at least one different unnatural amino acid.

Proteins or polypeptides of interest with at least one unnatural aminoacid are a feature of the invention. The invention also includespolypeptides or proteins with at least one unnatural amino acid producedusing the compositions and methods of the invention. An excipient(including but not limited to, a pharmaceutically acceptable excipient)can also be present with the protein.

By producing proteins or polypeptides of interest with at least oneunnatural amino acid in eukaryotic cells, proteins or polypeptides willtypically include eukaryotic post-translational modifications. Incertain embodiments, a protein includes at least one unnatural aminoacid and at least one post-translational modification that is made invivo by a eukaryotic cell, where the post-translational modification isnot made by a prokaryotic cell. For example, the post-translationmodification includes, including but not limited to, acetylation,acylation, lipid-modification, palmitoylation, palmitate addition,phosphorylation, glycolipid-linkage modification, glycosylation, and thelike. In one aspect, the post-translational modification includesattachment of an oligosaccharide (including but not limited to,(GlcNAc-Man)₂-Man-GlcNAc-GlcNAc)) to an asparagine by aGlcNAc-asparagine linkage. See Table 1 which lists some examples ofN-linked oligosaccharides of eukaryotic proteins (additional residuescan also be present, which are not shown). In another aspect, thepost-translational modification includes attachment of anoligosaccharide (including but not limited to, Gal-GalNAc, Gal-GlcNAc,etc.) to a serine or threonine by a GalNAc-serine or GalNAc-threoninelinkage, or a GlcNAc-serine or a GlcNAc-threonine linkage. TABLE 1EXAMPLES OF OLIGOSACCHARIDES THROUGH GlcNAc-LINKAGE Type Base StructureHigh- man- nose

Hybrid

Com- plex

Xylose

In yet another aspect, the post-translation modification includesproteolytic processing of precursors (including but not limited to,calcitonin precursor, calcitonin gene-related peptide precursor,preproparathyroid hormone, preproinsulin, proinsuliin,preproopiomelanocortin, pro-opiomelanocortin and the like), assemblyinto a multisubunit protein or macromolecular assembly, translation toanother site in the cell (including but not limited to, to organelles,such as the endoplasmic reticulum, the Golgi apparatus, the nucleus,lysosomes, peroxisomes, mitochondria, chloroplasts, vacuoles, etc., orthrough the secretory pathway). In certain embodiments, the proteincomprises a secretion or localization sequence, an epitope tag, a FLAGtag, a polyhistidine tag, a GST fusion, or the like. U.S. Pat. Nos.4,963,495 and 6,436,674, which are incorporated herein by reference,detail constructs designed to improve secretion of hGH polypeptides.

One advantage of an unnatural amino acid is that it presents additionalchemical moieties that can be used to add additional molecules. Thesemodifications can be made in vivo in a eukaryotic or non-eukaryoticcell, or in vitro. Thus, in certain embodiments, the post-translationalmodification is through the unnatural amino acid. For example, thepost-translational modification can be through anucleophilic-electrophilic reaction. Most reactions currently used forthe selective modification of proteins involve covalent bond formationbetween nucleophilic and electrophilic reaction partners, including butnot limited to the reaction of α-haloketones with histidine or cysteineside chains. Selectivity in these cases is determined by the number andaccessibility of the nucleophilic residues in the protein. In proteinsof the invention, other more selective reactions can be used such as thereaction of an unnatural keto-amino acid with hydrazides or aminooxycompounds, in vitro and in vivo. See, e.g., Cornish, et al., (1996) J.Am. Chem. Soc., 118:8150-8151; Mahal, et al., (1997) Science,276:1125-1128; Wang, et al., (2001) Science 292:498-500; Chin, et al.,(2002) J. Am. Chem. Soc. 124:9026-9027; Chin, et al., (2002) Proc. Natl.Acad. Sci., 99:11020-11024; Wang, et al., (2003) Proc. Natl. Acad. Sci.,100:56-61; Zhang, et al., (2003) Biochemistry, 42:6735-6746; and, Chin,et al., (2003) Science, 301:964-7, all of which are incorporated byreference herein. This allows the selective labeling of virtually anyprotein with a host of reagents including fluorophores, crosslinkingagents, saccharide derivatives and cytotoxic molecules. See also, U.S.Pat. No. 6,927,042 entitled “Glycoprotein synthesis,” which isincorporated by reference herein. Post-translational modifications,including but not limited to, through an azido amino acid, can also madethrough the Staudinger ligation (including but not limited to, withtriarylphosphine reagents). See, e.g., Kiick et al., (2002)Incorporation of azides into recombinant proteins for chemoselectivemodification by the Staudinger ligation, PNAS 99:19-24.

This invention provides another highly efficient method for theselective modification of proteins, which involves the geneticincorporation of unnatural amino acids, including but not limited to,containing an azide or alkynyl moiety into proteins in response to aselector codon. These amino acid side chains can then be modified by,including but not limited to, a Huisgen [3+2]cycloaddition reaction(see, e.g., Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991)Ed. Trost, B. M., Pergamon, Oxford, p. 1069-1109; and, Huisgen, R. in1,3-Dipolar Cycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, NewYork, p. 1-176) with, including but not limited to, alkynyl or azidederivatives, respectively. Because this method involves a cycloadditionrather than a nucleophilic substitution, proteins can be modified withextremely high selectivity. This reaction can be carried out at roomtemperature in aqueous conditions with excellent regioselectivity(1,4>1,5) by the addition of catalytic amounts of Cu(I) salts to thereaction mixture. See, e.g., Tomoe, et al., (2002) J. Org. Chem.67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed.41:2596-2599. Another method that can be used is the ligand exchange ona bisarsenic compound with a tetracysteine motif, see, e.g., Griffin, etal., (1998) Science 281:269-272.

A molecule that can be added to a protein of the invention through a[3+2]cycloaddition includes virtually any molecule with an azide oralkynyl derivative. Molecules include, but are not limited to, dyes,fluorophores, crosslinking agents, saccharide derivatives, polymers(including but not limited to, derivatives of polyethylene glycol),photocrosslinkers, cytotoxic compounds, affinity labels, derivatives ofbiotin, resins, beads, a second protein or polypeptide (or more),polynucleotide(s) (including but not limited to, DNA, RNA, etc.), metalchelators, cofactors, fatty acids, carbohydrates, and the like. Thesemolecules can be added to an unnatural amino acid with an alkynyl group,including but not limited to, p-propargyloxyphenylalanine, or azidogroup, including but not limited to, p-azido-phenylalanine,respectively.

V. In Vivo Generation of hIFN Polypeptides ComprisingNon-Genetically-Encoded Amino Acids

The hIFN polypeptides of the invention can be generated in vivo usingmodified tRNA and tRNA synthetases to add to or substitute amino acidsthat are not encoded in naturally-occurring systems.

Methods for generating tRNAs and tRNA synthetases which use amino acidsthat are not encoded in naturally-occurring systems are described in,e.g., U.S. Pat. No. 7,045,337 (Ser. No. 10/126,927) and U.S. PatentApplication Publication 2003/0108885 (Ser. No. 10/126,931) which areincorporated by reference herein. These methods involve generating atranslational machinery that functions independently of the synthetasesand tRNAs endogenous to the translation system (and are thereforesometimes referred to as “orthogonal”). Typically, the translationsystem comprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyltRNA synthetase (O-RS). Typically, the O-RS preferentially aminoacylatesthe O-tRNA with at least one non-naturally occurring amino acid in thetranslation system and the O-tRNA recognizes at least one selector codonthat is not recognized by other tRNAs in the system. The translationsystem thus inserts the non-naturally-encoded amino acid into a proteinproduced in the system, in response to an encoded selector codon,thereby “substituting” an amino acid into a position in the encodedpolypeptide.

A wide variety of orthogonal tRNAs and aminoacyl tRNA synthetases havebeen described in the art for inserting particular synthetic amino acidsinto polypeptides, and are generally suitable for use in the presentinvention. For example, keto-specific O-tRNA/aminoacyl-tRNA synthetasesare described in Wang, L., et al., Proc. Natl. Acad. Sci. USA 100:56-61(2003) and Zhang, Z. et al., Biochem. 42(22):6735-6746 (2003). ExemplaryO-RS, or portions thereof, are encoded by polynucleotide sequences andinclude amino acid sequences disclosed in U.S. Pat. No. 7,045,337 andU.S. Patent Application Publication 2003/0108885, each incorporatedherein by reference. Corresponding O-tRNA molecules for use with theO—RSs are also described in U.S. Pat. No. 7,045,337 (Ser. No.10/126,927) and U.S. Patent Application Publication 2003/0108885 (Ser.No. 10/126,931) which are incorporated by reference herein.

An example of an azide-specific O-tRNA/aminoacyl-tRNA synthetase systemis described in Chin, J. W., et al., J. Am. Chem., Soc. 124:9026-9027(2002). Exemplary O-RS sequences for p-azido-L-Phe include, but are notlimited to, nucleotide sequences SEQ ID NOs: 14-16 and 29-32 and aminoacid sequences SEQ ID NOs: 46-48 and 61-64 as disclosed in U.S. PatentApplication Publication 2003/0108885 (Ser. No. 10/126,931) which isincorporated by reference herein. Exemplary O-tRNA sequences suitablefor use in the present invention include, but are not limited to,nucleotide sequences SEQ ID NOs: 1-3 as disclosed in U.S. PatentApplication Publication 2003/0108885 (Ser. No. 10/126,931) which isincorporated by reference herein. Other examples ofO-tRNA/aminoacyl-tRNA synthetase pairs specific to particularnon-naturally encoded amino acids are described in U.S. Pat. No.7,045,337 (Ser. No. 10/126,927) which is incorporated by referenceherein. O-RS and O-tRNA that incorporate both keto- and azide-containingamino acids in S. cerevisiae are described in Chin, J. W., et al.,Science 301:964-967 (2003).

Several other orthogonal pairs have been reported. Glutaminyl (see,e.g., Liu, D. R., and Schultz, P. G. (1999) Proc. Natl. Acad. Sci.U.S.A. 96:4780-4785), aspartyl (see, e.g., Pastrnak, M., et al., (2000)Helv. Chim. Acta 83:2277-2286), and tyrosyl (see, e.g., Ohno, S., etal., (1998) J. Biochem. (Tokyo, Jpn.) 124:1065-1068; and, Kowal, A. K.,et al., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:2268-2273) systemsderived from S. cerevisiae tRNA's and synthetases have been describedfor the potential incorporation of unnatural amino acids in E. coli.Systems derived from the E. coli glutaminyl (see, e.g., Kowal, A. K., etal., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:2268-2273) and tyrosyl(see, e.g., Edwards, H., and Schimmel, P. (1990) Mol. Cell. Biol.10:1633-1641) synthetases have been described for use in S. cerevisiae.The E. coli tyrosyl system has been used for the incorporation of3-iodo-L-tyrosine in vivo, in mammalian cells. See, Sakamoto, K., etal., (2002) Nucleic Acids Res. 30:4692-4699.

Use of O-tRNA/aminoacyl-tRNA synthetases involves selection of aspecific codon which encodes the non-naturally encoded amino acid. Whileany codon can be used, it is generally desirable to select a codon thatis rarely or never used in the cell in which the O-tRNA/aminoacyl-tRNAsynthetase is expressed. For example, exemplary codons include nonsensecodon such as stop codons (amber, ochre, and opal), four or more basecodons and other natural three-base codons that are rarely or unused.

Specific selector codon(s) can be introduced into appropriate positionsin the hIFN polynucleotide coding sequence using mutagenesis methodsknown in the art (including but not limited to, site-specificmutagenesis, cassette mutagenesis, restriction selection mutagenesis,etc.).

Methods for generating components of the protein biosynthetic machinery,such as O-RSs, O-tRNAs, and orthogonal O-tRNA/O-RS pairs that can beused to incorporate a non-naturally encoded amino acid are described inWang, L., et al., Science 292: 498-500 (2001); Chin, J. W., et al., J.Am. Chem. Soc. 124:9026-9027 (2002); Zhang, Z. et al., Biochemistry 42:6735-6746 (2003). Methods and compositions for the in vivo incorporationof non-naturally encoded amino acids are described in U.S. Pat. No.7,045,337 (Ser. No. 10/126,927) which is incorporated by referenceherein. Methods for selecting an orthogonal tRNA-tRNA synthetase pairfor use in in vivo translation system of an organism are also describedin U.S. Pat. No. 7,045,337 (Ser. No. 10/126,927) and U.S. PatentApplication Publication 2003/0108885 (Ser. No. 10/126,931) which areincorporated by reference herein. PCT Publication No. WO 04/035743entitled “Site Specific Incorporation of Keto Amino Acids intoProteins,” which is incorporated by reference herein in its entirety,describes orthogonal RS and tRNA pairs for the incorporation of ketoamino acids. PCT Publication No. WO 04/094593 entitled “Expanding theEukaryotic Genetic Code,” which is incorporated by reference herein inits entirety, describes orthogonal RS and tRNA pairs for theincorporation of non-naturally encoded amino acids in eukaryotic hostcells.

Methods for producing at least one recombinant orthogonal aminoacyl-tRNAsynthetase (O-RS) comprise: (a) generating a library of (optionallymutant) RSs derived from at least one aminoacyl-tRNA synthetase (RS)from a first organism, including but not limited to, a prokaryoticorganism, such as Methanococcus jannaschii, Methanobacteriumthermoautotrophicum, Halobacterium, Escherichia coli, A. fulgidus, P.furiosus, P. horikoshii, A. pernix, T thermophilus, or the like, or aeukaryotic organism; (b) selecting (and/or screening) the library of RSs(optionally mutant RSs) for members that aminoacylate an orthogonal tRNA(O-tRNA) in the presence of a non-naturally encoded amino acid and anatural amino acid, thereby providing a pool of active (optionallymutant) RSs; and/or, (c) selecting (optionally through negativeselection) the pool for active RSs (including but not limited to, mutantRSs) that preferentially aminoacylate the O-tRNA in the absence of thenon-naturally encoded amino acid, thereby providing the at least onerecombinant O-RS; wherein the at least one recombinant O-RSpreferentially aminoacylates the O-tRNA with the non-naturally encodedamino acid.

In one embodiment, the RS is an inactive RS. The inactive RS can begenerated by mutating an active RS. For example, the inactive RS can begenerated by mutating at least about 1, at least about 2, at least about3, at least about 4, at least about 5, at least about 6, or at leastabout 10 or more amino acids to different amino acids, including but notlimited to, alanine.

Libraries of mutant RSs can be generated using various techniques knownin the art, including but not limited to rational design based onprotein three dimensional RS structure, or mutagenesis of RS nucleotidesin a random or rational design technique. For example, the mutant RSscan be generated by site-specific mutations, random mutations, diversitygenerating recombination mutations, chimeric constructs, rational designand by other methods described herein or known in the art.

In one embodiment, selecting (and/or screening) the library of RSs(optionally mutant RSs) for members that are active, including but notlimited to, that aminoacylate an orthogonal tRNA (O-tRNA) in thepresence of a non-naturally encoded amino acid and a natural amino acid,includes: introducing a positive selection or screening marker,including but not limited to, an antibiotic resistance gene, or thelike, and the library of (optionally mutant) RSs into a plurality ofcells, wherein the positive selection and/or screening marker comprisesat least one selector codon, including but not limited to, an amber,ochre, or opal codon; growing the plurality of cells in the presence ofa selection agent; identifying cells that survive (or show a specificresponse) in the presence of the selection and/or screening agent bysuppressing the at least one selector codon in the positive selection orscreening marker, thereby providing a subset of positively selectedcells that contains the pool of active (optionally mutant) RSs.Optionally, the selection and/or screening agent concentration can bevaried.

In one aspect, the positive selection marker is a chloramphenicolacetyltransferase (CAT) gene and the selector codon is an amber stopcodon in the CAT gene. Optionally, the positive selection marker is aβ-lactamase gene and the selector codon is an amber stop codon in theβ-lactamase gene. In another aspect the positive screening markercomprises a fluorescent or luminescent screening marker or an affinitybased screening marker (including but not limited to, a cell surfacemarker).

In one embodiment, negatively selecting or screening the pool for activeRSs (optionally mutants) that preferentially aminoacylate the O-tRNA inthe absence of the non-naturally encoded amino acid includes:introducing a negative selection or screening marker with the pool ofactive (optionally mutant) RSs from the positive selection or screeninginto a plurality of cells of a second organism, wherein the negativeselection or screening marker comprises at least one selector codon(including but not limited to, an antibiotic resistance gene, includingbut not limited to, a chloramphenicol acetyltransferase (CAT) gene);and, identifying cells that survive or show a specific screeningresponse in a first medium supplemented with the non-naturally encodedamino acid and a screening or selection agent, but fail to survive or toshow the specific response in a second medium not supplemented with thenon-naturally encoded amino acid and the selection or screening agent,thereby providing surviving cells or screened cells with the at leastone recombinant O-RS. For example, a CAT identification protocoloptionally acts as a positive selection and/or a negative screening indetermination of appropriate O-RS recombinants. For instance, a pool ofclones is optionally replicated on growth plates containing CAT (whichcomprises at least one selector codon) either with or without one ormore non-naturally encoded amino acid. Colonies growing exclusively onthe plates containing non-naturally encoded amino acids are thusregarded as containing recombinant O-RS. In one aspect, theconcentration of the selection (and/or screening) agent is varied. Insome aspects the first and second organisms are different. Thus, thefirst and/or second organism optionally comprises: a prokaryote, aeukaryote, a mammal, an Escherichia coli, a fungi, a yeast, anarchaebacterium, a eubactcrium, a plant, an insect, a protist, etc. Inother embodiments, the screening marker comprises a fluorescent orluminescent screening marker or an affinity based screening marker.

In another embodiment, screening or selecting (including but not limitedto, negatively selecting) the pool for active (optionally mutant) RSsincludes: isolating the pool of active mutant RSs from the positiveselection step (b); introducing a negative selection or screeningmarker, wherein the negative selection or screening marker comprises atleast one selector codon (including but not limited to, a toxic markergene, including but not limited to, a ribonuclease barnase gene,comprising at least one selector codon), and the pool of active(optionally mutant) RSs into a plurality of cells of a second organism;and identifying cells that survive or show a specific screening responsein a first medium not supplemented with the non-naturally encoded aminoacid, but fail to survive or show a specific screening response in asecond medium supplemented with the non-naturally encoded amino acid,thereby providing surviving or screened cells with the at least onerecombinant O-RS, wherein the at least one recombinant O-RS is specificfor the non-naturally encoded amino acid. In one aspect, the at leastone selector codon comprises about two or more selector codons. Suchembodiments optionally can include wherein the at least one selectorcodon comprises two or more selector codons, and wherein the first andsecond organism are different (including but not limited to, eachorganism is optionally, including but not limited to, a prokaryote, aeukaryote, a mammal, an Escherichia Coli, a fungi, a yeast, anarchaebacteria, a eubacteria, a plant, an insect, a protist, etc.).Also, some aspects include wherein the negative selection markercomprises a ribonuclease barnase gene (which comprises at least oneselector codon). Other aspects include wherein the screening markeroptionally comprises a fluorescent or luminescent screening marker or anaffinity based screening marker. In the embodiments herein, thescreenings and/or selections optionally include variation of thescreening and/or selection stringency.

In one embodiment, the methods for producing at least one recombinantorthogonal aminoacyl-tRNA synthetase (O-RS) can further comprise: (d)isolating the at least one recombinant O-RS; (e) generating a second setof O-RS (optionally mutated) derived from the at least one recombinantO-RS; and, (f) repeating steps (b) and (c) until a mutated O-RS isobtained that comprises an ability to preferentially aminoacylate theO-tRNA. Optionally, steps (d)-(f) are repeated, including but notlimited to, at least about two times. In one aspect, the second set ofmutated O-RS derived from at least one recombinant O-RS can be generatedby mutagenesis, including but not limited to, random mutagenesis,site-specific mutagenesis, recombination or a combination thereof.

The stringency of the selection/screening steps, including but notlimited to, the positive selection/screening step (b), the negativeselection/screening step (c) or both the positive and negativeselection/screening steps (b) and (c), in the above-described methods,optionally includes varying the selection/screening stringency. Inanother embodiment, the positive selection/screening step (b), thenegative selection/screening step (c) or both the positive and negativeselection/screening steps (b) and (c) comprise using a reporter, whereinthe reporter is detected by fluorescence-activated cell sorting (FACS)or wherein the reporter is detected by luminescence. Optionally, thereporter is displayed on a cell surface, on a phage display or the likeand selected based upon affinity or catalytic activity involving thenon-naturally encoded amino acid or an analogue. In one embodiment, themutated synthetase is displayed on a cell surface, on a phage display orthe like.

Methods for producing a recombinant orthogonal tRNA (O-tRNA) include:(a) generating a library of mutant tRNAs derived from at least one tRNA,including but not limited to, a suppressor tRNA, from a first organism;(b) selecting (including but not limited to, negatively selecting) orscreening the library for (optionally mutant) tRNAs that areaminoacylated by an aminoacyl-tRNA synthetase (RS) from a secondorganism in the absence of a RS from the first organism, therebyproviding a pool of tRNAs (optionally mutant); and, (c) selecting orscreening the pool of tRNAs (optionally mutant) for members that areaminoacylated by an introduced orthogonal RS(O-RS), thereby providing atleast one recombinant O-tRNA; wherein the at least one recombinantO-tRNA recognizes a selector codon and is not efficiency recognized bythe RS from the second organism and is preferentially aminoacylated bythe O-RS. In some embodiments the at least one tRNA is a suppressor tRNAand/or comprises a unique three base codon of natural and/or unnaturalbases, or is a nonsense codon, a rare codon, an unnatural codon, a codoncomprising at least 4 bases, an amber codon, an ochre codon, or an opalstop codon. In one embodiment, the recombinant O-tRNA possesses animprovement of orthogonality. It will be appreciated that in someembodiments, O-tRNA is optionally imported into a first organism from asecond organism without the need for modification. In variousembodiments, the first and second organisms are either the same ordifferent and are optionally chosen from, including but not limited to,prokaryotes (including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Escherichia coli, Halobacterium,etc.), eukaryotes, mammals, fungi, yeasts, archaebacteria, eubacteria,plants, insects, protists, etc. Additionally, the recombinant tRNA isoptionally aminoacylated by a non-naturally encoded amino acid, whereinthe non-naturally encoded amino acid is biosynthesized in vivo eithernaturally or through genetic manipulation. The non-naturally encodedamino acid is optionally added to a growth medium for at least the firstor second organism.

In one aspect, selecting (including but not limited to, negativelyselecting) or screening the library for (optionally mutant) tRNAs thatare aminoacylated by an aminoacyl-tRNA synthetase (step (b)) includes:introducing a toxic marker gene, wherein the toxic marker gene comprisesat least one of the selector codons (or a gene that leads to theproduction of a toxic or static agent or a gene essential to theorganism wherein such marker gene comprises at least one selector codon)and the library of (optionally mutant) tRNAs into a plurality of cellsfrom the second organism; and, selecting surviving cells, wherein thesurviving cells contain the pool of (optionally mutant) tRNAs comprisingat least one orthogonal tRNA or nonfunctional tRNA. For example,surviving cells can be selected by using a comparison ratio cell densityassay.

In another aspect, the toxic marker gene can include two or moreselector codons. In another embodiment of the methods, the toxic markergene is a ribonuclease barnase gene, where the ribonuclease barnase genecomprises at least one amber codon. Optionally, the ribonuclease barnasegene can include two or more amber codons.

In one embodiment, selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS(O-RS) can include: introducing a positive selection orscreening marker gene, wherein the positive marker gene comprises a drugresistance gene (including but not limited to, β-lactamase gene,comprising at least one of the selector codons, such as at least oneamber stop codon) or a gene essential to the organism, or a gene thatleads to detoxification of a toxic agent, along with the O-RS, and thepool of (optionally mutant) tRNAs into a plurality of cells from thesecond organism; and, identifying surviving or screened cells grown inthe presence of a selection or screening agent, including but notlimited to, an antibiotic, thereby providing a pool of cells possessingthe at least one recombinant tRNA, where the at least one recombinanttRNA is aminoacylated by the O-RS and inserts an amino acid into atranslation product encoded by the positive marker gene, in response tothe at least one selector codons. In another embodiment, theconcentration of the selection and/or screening agent is varied.

Methods for generating specific O-tRNA/O-RS pairs are provided. Methodsinclude: (a) generating a library of mutant tRNAs derived from at leastone tRNA from a first organism; (b) negatively selecting or screeningthe library for (optionally mutant) tRNAs that are aminoacylated by anaminoacyl-tRNA synthetase (RS) from a second organism in the absence ofa RS from the first organism, thereby providing a pool of (optionallymutant) tRNAs; (c) selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS(O-RS), thereby providing at least one recombinant O-tRNA.The at least one recombinant O-tRNA recognizes a selector codon and isnot efficiency recognized by the RS from the second organism and ispreferentially aminoacylated by the O-RS. The method also includes (d)generating a library of (optionally mutant) RSs derived from at leastone aminoacyl-tRNA synthetase (RS) from a third organism; (e) selectingor screening the library of mutant RSs for members that preferentiallyaminoacylate the at least one recombinant O-tRNA in the presence of anon-naturally encoded amino acid and a natural amino acid, therebyproviding a pool of active (optionally mutant) RSs; and, (f) negativelyselecting or screening the pool for active (optionally mutant) RSs thatpreferentially aminoacylate the at least one recombinant O-tRNA in theabsence of the non-naturally encoded amino acid, thereby providing theat least one specific O-tRNA/O-RS pair, wherein the at least onespecific O-tRNA/O-RS pair comprises at least one recombinant O-RS thatis specific for the non-naturally encoded amino acid and the at leastone recombinant O-tRNA. Specific O-tRNA/O—RS pairs produced by themethods are included. For example, the specific O-tRNA/O—RS pair caninclude, including but not limited to, a mutRNATyr-mutTyrRS pair, suchas a mutRNATyr-SS12TyrRS pair, a mutRNALeu-mutLeuRS pair, amutRNAThr-mutThrRS pair, a mutRNAGlu-mutGluRS pair, or the like.Additionally, such methods include wherein the first and third organismare the same (including but not limited to, Methanococcus jannaschii).

Methods for selecting an orthogonal tRNA-tRNA synthetase pair for use inan in vivo translation system of a second organism are also included inthe present invention. The methods include: introducing a marker gene, atRNA and an aminoacyl-tRNA synthetase (RS) isolated or derived from afirst organism into a first set of cells from the second organism;introducing the marker gene and the tRNA into a duplicate cell set froma second organism; and, selecting for surviving cells in the first setthat fail to survive in the duplicate cell set or screening for cellsshowing a specific screening response that fail to give such response inthe duplicate cell set, wherein the first set and the duplicate cell setare grown in the presence of a selection or screening agent, wherein thesurviving or screened cells comprise the orthogonal tRNA-tRNA synthetasepair for use in the in the in vivo translation system of the secondorganism. In one embodiment, comparing and selecting or screeningincludes an in vivo complementation assay. The concentration of theselection or screening agent can be varied.

The organisms of the present invention comprise a variety of organismand a variety of combinations. For example, the first and the secondorganisms of the methods of the present invention can be the same ordifferent. In one embodiment, the organisms are optionally a prokaryoticorganism, including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, P. furiosus, P. horikoshii, A. pernix, T. thermophilus, orthe like. Alternatively, the organisms optionally comprise a eukaryoticorganism, including but not limited to, plants (including but notlimited to, complex plants such as monocots, or dicots), algae,protists, fungi (including but not limited to, yeast, etc), animals(including but not limited to, mammals, insects, arthropods, etc.), orthe like. In another embodiment, the second organism is a prokaryoticorganism, including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, Halobacterium, P. furiosus, P. horikoshii, A. pernix, T.thermophilus, or the like. Alternatively, the second organism can be aeukaryotic organism, including but not limited to, a yeast, a animalcell, a plant cell, a fungus, a mammalian cell, or the like. In variousembodiments the first and second organisms are different.

VI. Location of Non-Naturally-Occurring Amino Acids in hIFN Polypeptides

The present invention contemplates incorporation of one or morenon-naturally-occurring amino acids into hIFN polypeptides. One or morenon-naturally-occurring amino acids may be incorporated at a particularposition which does not disrupt activity of the polypeptide. This can beachieved by making “conservative” substitutions, including but notlimited to, substituting hydrophobic amino acids with hydrophobic aminoacids, bulky amino acids for bulky amino acids, hydrophilic amino acidsfor hydrophilic amino acids and/or inserting the non-naturally-occurringamino acid in a location that is not required for activity.

Regions of hIFN can be illustrated as follows, wherein the amino acidpositions in hIFN are according to SEQ ID NO:2:

1-9 (N-terminus), 10-21 (A helix), 22-39 (region between A helix and Bhelix), 40-75 (B helix), 76-77 (region between B helix and C helix),78-100 (C helix), 101-110 (region between C helix and D helix), 111-132(D helix), 133-136 (region between D and E helix) 137-155 (E helix)156-165 (C-terminus).

A variety of biochemical and structural approaches can be employed toselect the desired sites for substitution with a non-naturally encodedamino acid within the hIFN polypeptide. It is readily apparent to thoseof ordinary skill in the art that any position of the polypeptide chainis suitable for selection to incorporate a non-naturally encoded aminoacid, and selection may be based on rational design or by randomselection for any or no particular desired purpose. Selection of desiredsites may be for producing a hIFN molecule having any desired propertyor activity, including but not limited to, agonists, super-agonists,inverse agonists, antagonists, receptor binding modulators, receptoractivity modulators, dimer or multimer formation, no change to activityor property compared to the native molecule, or manipulating anyphysical or chemical property of the polypeptide such as solubility,aggregation, or stability. For example, locations in the polypeptiderequired for biological activity of hIFN polypeptides can be identifiedusing point mutation analysis, alanine scanning or homolog scanningmethods known in the art. See, e.g., Di Marco et al., Biochem BiophysRes Corn 202:1445 (1994); Walter et al., Cancer Biotherapy & Radiopharm.13:143 (1998); Runkel et al., J.B.C. 273:8003 (1998) for IFN. U.S. Pat.Nos. 5,580,723; 5,834,250; 6,013,478; 6,428,954; and 6,451,561, whichare incorporated by reference herein, describe methods for thesystematic analysis of the structure and function of polypeptides suchas hGH by identifying active domains which influence the activity of thepolypeptide with a target substance. Residues other than thoseidentified as critical to biological activity by alanine or homologscanning mutagenesis may be good candidates for substitution with anon-naturally encoded amino acid depending on the desired activitysought for the polypeptide. Alternatively, the sites identified ascritical to biological activity may also be good candidates forsubstitution with a non-naturally encoded amino acid, again depending onthe desired activity sought for the polypeptide. Another alternativewould be to simply make serial substitutions in each position on thepolypeptide chain with a non-naturally encoded amino acid and observethe effect on the activities of the polypeptide. Alternatively, residuesthat modulate one or more of the biological activities of IFN includingside effects found with current IFN therapeutics may be good candidatesfor substitution with a non-naturally encoded amino acid. Such sideeffects include but are not limited to, neutropenia. Alternatively,residues that modulate toxicity may be good candidates for substitutionwith a non-naturally encoded amino acid. Such residues may include, butare not limited to, residues that interact with one or more members ofthe opioid family of receptors and/or residues to modulate the inductionof indoleamine 2,3-dioxygenase. Residues that improve antiviral activitymay be good candidates for substitution with a non-naturally encodedamino acid. Chimeric hIFN polypeptides may be designed incorporatingregions or sites from limitin into IFNα to provide an improved hIFNpolypeptide with retained or improved antiviral activity and/ormodulated side effects and/or modulated toxicity. One or more residuesin hIFN may be substituted with one or more residues found in limitin.One or more residues in the C-D loop may be good candidates forsubstitution with a non-naturally encoded amino acid or othermodifications, including but not limited to, substitution of residues inIFN with residues found in limitin. A majority of the residues in theC-D loop of hIFN may be substituted with residues found in limitin.Suitable residues from limitin for substitution into hIFN may bedetermined by performing a comparison between the sequences,three-dimensional structure, secondary structure, one or more biologicalactivities, or receptor binding of limitin and hIFN. It is readilyapparent to those of ordinary skill in the art that any means,technique, or method for selecting a position for substitution with anon-natural amino acid into any polypeptide is suitable for use in thepresent invention.

The structure and activity of naturally-occurring mutants of hIFNpolypeptides that contain deletions can also be examined to determineregions of the protein that are likely to be tolerant of substitutionwith a non-naturally encoded amino acid. In a similar manner, proteasedigestion and monoclonal antibodies can be used to identify regions ofhIFN that are responsible for binding the hIFN receptor. Once residuesthat are likely to be intolerant to substitution with non-naturallyencoded amino acids have been eliminated, the impact of proposedsubstitutions at each of the remaining positions can be examined fromthe three-dimensional crystal structure of the hIFN and its bindingproteins. X-ray crystallographic and NMR structures of hIFN are alsoavailable in the Protein Data Bank (including 1RH2 and 1ITF) (PDB,available on the World Wide Web at rcsb.org), a centralized databasecontaining three-dimensional structural data of large molecules ofproteins and nucleic acids, as well as U.S. Pat. Nos. 5,602,232;5,460,956; 5,441,734; 4,672,108, which are incorporated by referenceherein. Models may be made investigating the secondary and tertiarystructure of polypeptides, if three-dimensional structural data is notavailable. Thus, those of ordinary skill in the art can readily identifyamino acid positions that can be substituted with non-naturally encodedamino acids.

In some embodiments, the hIFN polypeptides of the invention comprise oneor more non-naturally occurring amino acids positioned in a region ofthe protein that does not disrupt the helices or beta sheet secondarystructure of the polypeptide.

Exemplary residues of incorporation of a non-naturally encoded aminoacid may be those that are excluded from potential receptor bindingregions (including but not limited to, Site I and Site II), may be fullyor partially solvent exposed, have minimal or no hydrogen-bondinginteractions with nearby residues, may be minimally exposed to nearbyreactive residues, and may be in regions that are highly flexible(including but not limited to, C-D loop) or structurally rigid(including but not limited to, B helix) as predicted by thethree-dimensional crystal structure, secondary, tertiary, or quaternarystructure of the hIFN polypeptide, bound or unbound to its receptor.Residues of incorporation of a non-naturally encoded amino acid may bethose that modulate one or more of the biological activities of IFNincluding side effects found with current IFN therapeutics. Residues ofincorporation of a non-naturally encoded amino acid may be those thatmodulate toxicity. Such residues include, but are not limited to,residues that are involved with binding to one or more members of theopioid receptor family and/or residues that modulate induction ofindoleamine 2,3-dioxygenase.

In some embodiments, one or more non-naturally encoded amino acid areincorporated or substituted in one or more of the following regionscorresponding to secondary structures in IFN wherein the amino acidpositions in hIFN are according to SEQ ID NO: 2:

1-9 (N-terminus), 10-21 (A helix), 22-39 (region between A helix and Bhelix), 40-75 (B helix), 76-77 (region between B helix and C helix),78-100 (C helix), 101-110 (region between C helix and D helix), 111-132(D helix), 133-136 (region between D and E helix) 137-155 (E helix)156-165 (C-terminus).

In some embodiments, one or more non-naturally encoded amino acid aresubstituted at, but not limited to, one or more of the followingpositions of hIFN (as in SEQ ID NO: 2, or the corresponding amino acidin SEQ ID NO: 1, 3, or any other IFN polypeptide): before position 1(i.e., at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, or 166 (i.e. at the carboxylterminus). In some embodiments, one or more non-naturally encoded aminoacids are incorporated in one or more of the following positions in IFN:before position 1 (i.e. at the N terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9,12, 13, 16, 19, 20, 22, 23, 24, 25, 26, 27, 28, 30, 31, 32, 33, 34, 35,40, 41, 42, 45, 46, 48, 49, 50, 51, 58, 61, 64, 65, 68, 69, 70, 71, 73,74, 77, 78, 79, 80, 81, 82, 83, 85, 86, 89, 90, 93, 94, 96, 97, 100,101, 103, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 117, 118,120, 121, 124, 125, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137,148, 149, 152, 153, 156, 158, 159, 160, 161, 162, 163, 164, 165, 166(i.e. at the carboxyl terminus of the protein) (as in SEQ ID NO: 2, orthe corresponding amino acids in other IFN's). In some embodiments, oneor more non-naturally encoded amino acids are incorporated in one ormore of the following positions in IFN: 6, 9, 12, 13, 16, 41, 45, 46,48, 49, 61, 64, 65, 96, 100, 101, 103, 106, 107, 108, 110, 111, 113,114, 117, 120, 121, 149, 156, 159, 160, 161 and 162 (SEQ ID NO: 2, orthe corresponding amino acids in SEQ ID NO: 1 or 3). In someembodiments, the IFN polypeptides of the invention comprise one or morenon-naturally encoded amino acids at one or more of the followingpositions: 100, 106, 107, 108, 111, 113, 114 (SEQ ID NO: 2, or thecorresponding amino acids in other IFN's). In some embodiments, the IFNpolypeptides of the invention comprise one or more non-naturally encodedamino acids at one or more of the following positions: 41, 45, 46, 48,49 (SEQ ID NO: 2, or the corresponding amino acids in other IFN's). Insome embodiments, the IFN polypeptides of the invention comprise one ormore non-naturally encoded amino acids at one or more of the followingpositions: 61, 64, 65, 101, 103, 110, 117, 120, 121, 149 (SEQ ID NO: 2,or the corresponding amino acids in other IFN's). In some embodiments,the IFN polypeptides of the invention comprise one or more non-naturallyencoded amino acids at one or more of the following positions: 6, 9, 12,13, 16, 96, 156, 159, 160, 161, 162 (SEQ ID NO: 2, or the correspondingamino acids in other IFN's). In some embodiments, the IFN polypeptidesof the invention comprise one or more non-naturally occurring aminoacids at one or more of the following positions: 2, 3, 4, 5, 7, 8, 16,19, 20, 40, 42, 50, 51, 58, 68, 69, 70, 71, 73, 97, 105, 109, 112, 118,148, 149, 152, 153, 158, 163, 164, 165 (SEQ ID NO: 2, or thecorresponding amino acids in other IFN's). In some embodiments, the IFNpolypeptides of the invention comprise one or more non-naturally encodedamino acids at one or more of the following positions: 34, 78, 107 (SEQID NO: 2, or the corresponding amino acid in SEQ ID NO: 1, 3, or anyother IFN polypeptide). In some embodiments, the non-naturally encodedamino acid at one or more of these or other positions is linked to awater soluble polymer, including but not limited to positions: beforeposition 1 (i.e., at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, or 166 (i.e. at thecarboxyl terminus) (SEQ ID NO: 2, or the corresponding amino acid in SEQID NO: 1, 3, or any other IFN polypeptide). In some embodiments, thenon-naturally encoded amino acid at these or other positions is linkedto a water soluble polymer, including but not limited to positions:before position 1 (i.e. the N terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 12,13, 16, 19, 20, 22, 23, 24, 25, 26, 27, 28, 30, 31, 32, 33, 34, 35, 40,41, 42, 45, 46, 48, 49, 50, 51, 58, 61, 64, 65, 68, 69, 70, 71, 73, 74,77, 78, 79, 80, 81, 82, 83, 85, 86, 89, 90, 93, 94, 96, 97, 100, 101,103, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 117, 118, 120,121, 124, 125, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137, 148,149, 152, 153, 156, 158, 159, 160, 161, 162, 163, 164, 165, 166 (i.e.the carboxyl terminus) (SEQ ID NO: 2, or the corresponding amino acidsin other IFN's). In some embodiments, the water soluble polymer iscoupled at one or more amino acid positions: 6, 9, 12, 13, 16, 41, 45,46, 48, 49, 61, 64, 65, 96, 100, 101, 103, 106, 107, 108, 110, 111, 113,114, 117, 120, 121, 149, 156, 159, 160, 161 and 162 (SEQ ID NO: 2, orthe corresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide). In some embodiments, the non-naturally encoded amino acidis linked to a water soluble polymer at one or more of the followingpositions: 100, 106, 107, 108, 111, 113, 114 (SEQ ID NO: 2, or thecorresponding amino acids in SEQ ID NO: 1 or 3). In some embodiments,the non-naturally encoded amino acid is linked to a water solublepolymer at one or more of the following positions: 41, 45, 46, 48, 49(SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 1 or 3).In some embodiments, the non-naturally encoded amino acid is linked to awater soluble polymer at one or more of the following positions: 61, 64,65, 101, 103, 110, 117, 120, 121, 149 (SEQ ID NO: 2, or thecorresponding amino acids in SEQ ID NO: 1 or 3). In some embodiments,the non-naturally encoded amino acid is linked to a water solublepolymer at one or more of the following positions: 6, 9, 12, 13, 16, 96,156, 159, 160, 161, 162 (SEQ ID NO: 2, or the corresponding amino acidsin SEQ ID NO: 1 or 3).

In some embodiments, the one or more non-naturally encoded amino acidsat one or more of the following positions is linked to one or morewater-soluble polymer: 2, 3, 4, 5, 7, 8, 16, 19, 20, 40, 42, 50, 51, 58,68, 69, 70, 71, 73, 97, 105, 109, 112, 118, 148, 149, 152, 153, 158,163, 164, 165 (SEQ ID NO: 2, or the corresponding amino acid in or thecorresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide). In some embodiments, the one or more non-naturally encodedamino acids at one or more of the following positions is linked to oneor more water-soluble polymer: 34, 78, 107 (SEQ ID NO: 2, or thecorresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide). In some embodiments, the water soluble polymer is coupledto the IFN polypeptide to a non-naturally encoded amino acid at one ormore of the following amino acid positions: 6, 9, 12, 13, 16, 41, 45,46, 48, 49, 61, 64, 65, 96, 100, 101, 103, 106, 107, 108, 110, 111, 113,114, 117, 120, 121, 149, 156, 159, 160, 161 and 162 (SEQ ID NO: 2, orthe corresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide). In some embodiments the water soluble polymer is coupledto the IFN polypeptide at one or more of the following amino acidpositions: 6, 9, 12, 13, 16, 41, 45, 46, 48, 49, 61, 64, 65, 96, 100,101, 103, 106, 107, 108, 110, 111, 113, 114, 117, 120, 121, 149, 156,159, 160, 161 and 162 (SEQ ID NO: 2, or the corresponding amino acid inSEQ ID NO: 1, 3, or any other IFN polypeptide). In some embodiments, thenon-naturally encoded amino acid at one or more of these positions islinked to one or more water soluble polymers, positions: 34, 78, 107(SEQ ID NO: 2, or the corresponding amino acid in or the correspondingamino acid in SEQ ID NO: 1, 3, or any other IFN polypeptide). In someembodiments, the IFN polypeptides of the invention comprise one or morenon-naturally encoded amino acids at one or more of the followingpositions providing an antagonist: 2, 3, 4, 5, 7, 8, 16, 19, 20, 40, 42,50, 51, 58, 68, 69, 70, 71, 73, 97, 105, 109, 112, 118, 148, 149, 152,153, 158, 163, 164, 165 (SEQ ID NO: 2, or the corresponding amino acidin or the corresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide); a hIFN polypeptide comprising one of these substitutionsmay potentially act as a weak antagonist or weak agonist depending onthe intended site selected and desired activity. Human IFN antagonistsinclude, but are not limited to, hIFN polypeptides with one or morenon-naturally encoded amino acid substitutions at 22, 23, 24, 25, 26,27, 28, 30, 31, 32, 33, 34, 35, 74, 77, 78, 79, 80, 82, 83, 85, 86, 89,90, 93, 94, 124, 125, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137,or any combinations thereof (hIFN; SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NO: 1 or 3, or any other IFN polypeptide).

In some embodiments, one or more non-naturally encoded amino acids aresubstituted at, but not limited to, one or more of the followingpositions of hIFN (as in SEQ ID NO: 2, or the corresponding amino acidsin SEQ ID NO: 1 or 3, or any other IFN polypeptide): 31, 134, 34, 38,129, 36, 122, 37, 121, 41, 125, 124, 149, 117, 39, 118, 120, 107, 108,106, 100, 111, 113, 114, 41, 45, 46, 48, 49, 61, 64, 65, 101, 103, 102,110, 117, 120, 121, 149, 96, 6, 9, 12, 13, 16, 68, 70, 109, 159, 161,156, 160, 162, 24, 27, 78, 83, 85, 87, 89, 164. In one embodiment, anon-naturally encoded amino acid is substituted at position 38 of hIFN(as in SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 1 or3, or any other IFN polypeptide). In some embodiments, the non-naturallyencoded amino acid at one or more of these or other positions is linkedto a water soluble polymer, including but not limited to positions: 31,134, 34, 38, 129, 36, 122, 37, 121, 41, 125, 124, 149, 117, 39, 118,120, 107, 108, 106, 100, 111, 113, 114, 41, 45, 46, 48, 49, 61, 64, 65,101, 103, 102, 110, 117, 120, 121, 149, 96, 6, 9, 12, 13, 16, 68, 70,109, 159, 161, 156, 160, 162, 24, 27, 78, 83, 85, 87, 89, 164 (as in SEQID NO: 2, or the corresponding amino acids in SEQ ID NO: 1 or 3, or anyother IFN polypeptide).

In some embodiments, one or more non-naturally encoded amino acids aresubstituted at, but not limited to, one or more of the followingpositions of hIFN (SEQ ID NO: 2 or the corresponding amino acids in SEQID NO: 1 or 3): before position 1 (i.e., at the N-terminus), 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, or 166(i.e. at the carboxyl terminus) and one or more natural amino acidsubstitutions. In some embodiments, the one or more non-naturallyencoded amino acids are coupled to a water soluble polymer. In someembodiments, the one or more non-naturally encoded amino acids arecoupled to PEG. In one embodiment, the natural amino acid substitutionis R149Y. In some embodiments, the natural amino acid substitution isR149E. In some embodiments, the natural amino acid substitution isR149S. In one embodiment, the non-natural amino acid substitution is atposition 107 and the natural amino acid substitution is R149Y. In oneembodiment, the non-natural amino acid substitution is at position 106and the natural amino acid substitution is R149Y. In some embodiments,the one or more naturally encoded amino acid substitution is at one ormore of the following positions of hIFN (SEQ ID NO: 2 or thecorresponding amino acids in SEQ ID NO: 1 or 3), including but notlimited to: 10, 16, 13, 79, 83, 85, 86, 87, 90, 91, 93, 94, 96, 120,121, 124, 125, 128, 149. In some embodiments, the one or more naturallyencoded amino acid substitution is one or more of the followingsubstitutions (SEQ ID NO: 2 or the corresponding amino acids in SEQ IDNO: 1 or 3), including but not limited to: G10E, M16R, R13E, T79R, K83Q,K83S, Y85L, T86S, E87S, Q90R, Q91E, N93Q, D94V, E96K, R120K, K121T,Q124R, R125G, L128R, R149Y, R149E, R149S. In some embodiments, thenatural amino acid substitution is at position 1 (the N-terminus). Insome embodiments, one or more non-naturally encoded amino acids aresubstituted at one or more of the following positions of hIFN (as in SEQID NO: 2, or the corresponding amino acids in other IFN's): 107, 78, 34.In some embodiments, the non-naturally encoded amino acid at one or moreof these positions is coupled to a water soluble polymer: 107, 78, 34.

One or more amino acids found in a limitin sequence may be substitutedinto a hIFN polypeptide (hybrid limitin/hIFN polypeptides). Examplesinclude but are not limited to the natural amino acid substitutionsdescribed in the previous paragraph. Alternatively, a set of amino acidsfound in an interferon polypeptide may be replaced by a set of aminoacids found in a limitin sequence. A set of amino acids may comprisecontiguous amino acids or amino acids present in different portions ofthe molecule but are involved in a structural characteristic orbiological activity of the polypeptide. The mouse limitin molecule hasan improved CFU-GM toxicity profile compared to other IFNα proteins.Alignment of human IFNα-2a with the limitin protein sequence showed 30%amino acid identity. 50% sequence conservation was also observed. Inparticular, a prominent deletion in the limitin sequence between the Cand D helices (in the loop between C and D helices) was observed. The“HV” mutant was generated with the following substitutions in hIFNα-2a(SEQ ID NO: 2): D77-D94 is replaced with the mouse limitin sequenceHERALDQLLSSLWRELQV. The “CD” mutant was generated with the followingsubstitutions in hIFNα-2a (SEQ ID NO: 2): V105-D114 with GQSAPLP. Thishybrid molecule with the loop region from limitin substituted into thehuman IFNα-2a protein (“CD” mutant) was found to have equivalentanti-viral activity as the WHO IFN standard. In addition to the one ormore limitin amino acids, the hIFN polypeptide may comprise one or morenon-naturally encoded amino acids at any one or more positions of thehIFN polypeptide. In some embodiments, the one or more non-naturallyencoded amino acids may be linked to a water soluble polymer such as PEGor bonded directly to a water soluble polymer such as PEG. In additionto the natural amino acid substitutions in the HV or the CD mutant, oneor more additional natural amino acid substitutions may be found in thehIFN polypeptide.

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 6, 16, 34, 39, 45, 46, 64, 65, 68, 78, 85, 87, 101, 107, 108,111, 114, 118, 124, 125, 145, 146, 153, 156, 96, 149 (SEQ ID NO: 2, orthe corresponding amino acid in or the corresponding amino acid in SEQID NO: 1, 3, or any other IFN polypeptide). In some embodiments, thehIFN polypeptide comprises one or more non-naturally encoded amino acidsat one or more of the following positions linked to one or morewater-soluble polymer: 6, 16, 34, 39, 45, 46, 64, 65, 68, 78, 85, 87,101, 107, 108, 111, 114, 118, 124, 125, 145, 146, 153, 156, 96, 149 (SEQID NO: 2, or the corresponding amino acid in or the corresponding aminoacid in SEQ ID NO: 1, 3, or any other IFN polypeptide). In someembodiments, the hIFN polypeptide comprises one or more non-naturallyencoded amino acids at one or more of the following positions linked toone or more water-soluble polymer: 6, 16, 34, 39, 45, 46, 64, 65, 68,78, 85, 87, 101, 107, 108, 111, 114, 118, 124, 125, 145, 146, 153, 156,96, 149 (SEQ ID NO: 2, or the corresponding amino acid in or thecorresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide) and comprises one or more naturally encoded amino acidsubstitution. In some embodiments, the hIFN polypeptide comprises one ormore non-naturally encoded amino acids at one or more of the followingpositions linked to one or more water-soluble polymer: 6, 16, 34, 39,45, 46, 64, 65, 68, 78, 85, 87, 101, 107, 108, 111, 114, 118, 124, 125,145, 146, 153, 156, 96, 149 (SEQ ID NO: 2, or the corresponding aminoacid in or the corresponding amino acid in SEQ ID NO: 1, 3, or any otherIFN polypeptide) and comprises one or more of the following naturallyencoded amino acid substitutions G10E, M16R, R13E, T79R, K83Q, K83S,Y85L, T86S, E87S, Q90R, Q91E, N93Q, D94V, E96K, R120K, K121T, Q124R,R125G, L128R, R149Y, R149E, R149S.

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 6, 16, 37, 45, 46, 78, 87, 89, 107, 108 (SEQ ID NO: 2 or thecorresponding amino acids in SEQ ID NO: 1 or 3). In some embodiments,the hIFN polypeptide comprises one or more non-naturally encoded aminoacids at one or more of the following positions: 6, 16, 37, 45, 46, 78,87, 89, 107, 108 (SEQ ID NO: 2 or the corresponding amino acids in SEQID NO: 1 or 3) that is linked to a water soluble polymer. In someembodiments, the hIFN polypeptide comprises one or more non-naturallyencoded amino acids at one or more of the following positions: 6, 16,37, 45, 46, 78, 87, 89, 107, 108 (SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NO: 1 or 3) that is bonded to a water solublepolymer.

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 6, 16, 37, 45, 46, 78, 87, 89, 107, 108 and one or more ofthe following naturally encoded amino acid substitutions: T79R, L80A,K83S, Y85L, Y85S, T86S, E87S, Q91E (SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NO: 1 or 3). In some embodiments, the hIFNpolypeptide comprises one or more non-naturally encoded amino acids atone or more of the following positions: 6, 16, 37, 45, 46, 78, 87, 89,107, 108 that is linked to a water soluble polymer and comprises one ormore of the following naturally encoded amino acid substitutions: T79R,L80A, K83S, Y8511, Y85S, T86S, E87S, Q91E (SEQ ID NO: 2 or thecorresponding amino acids in SEQ ID NO: 1 or 3). In some embodiments,the hIFN polypeptide comprises one or more non-naturally encoded aminoacids at one or more of the following positions: 6, 16, 37, 45, 46, 78,87, 89, 107, 108 that is bonded to a water soluble polymer and comprisesone or more of the following naturally encoded amino acid substitutions:T79R, L80A, K83S, Y85L, Y85S, T86S, E87S, Q91E (SEQ ID NO: 2 or thecorresponding amino acids in SEQ ID NO: 1 or 3).

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 6, 16, 37, 45, 46, 78, 87, 107, and 108 (SEQ ID NO: 2 or thecorresponding amino acids in SEQ ID NO: 1 or 3). In some embodiments,the hIFN polypeptide comprises one or more non-naturally encoded aminoacids at one or more of the following positions: 6, 16, 37, 45, 46, 78,87, 107, and 108 (SEQ ID NO: 2 or the corresponding amino acids in SEQID NO: 1 or 3) that is linked to a water soluble polymer. In someembodiments, the hIFN polypeptide comprises one or more non-naturallyencoded amino acids at one or more of the following positions: 6, 16,37, 45, 46, 78, 87, 107, and 108 (SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NO: 1 or 3) that is bonded to a water solublepolymer.

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 6, 16, 37, 45, 46, 78, 87, 107, and 108 and comprises one ormore of the following naturally encoded amino acid substitutions: T79R,K83S, Y85L, T86S, E87S, Q91E (SEQ ID NO: 2 or the corresponding aminoacids in SEQ ID NO: 1 or 3). In some embodiments, the hIFN polypeptidecomprises one or more non-naturally encoded amino acids at one or moreof the following positions: 6, 16, 37, 45, 46, 78, 87, 107, and 108 thatis linked to a water soluble polymer and comprises one or more of thefollowing naturally encoded amino acid substitutions: T79R, K83S, Y85L,T86S, E87S, Q91E (SEQ ID NO: 2 or the corresponding amino acids in SEQID NO: 1 or 3). In some embodiments, the hIFN polypeptide comprises oneor more non-naturally encoded amino acids at one or more of thefollowing positions: 6, 16, 37, 45, 46, 78, 87, 107, and 108 that isbonded to a water soluble polymer and comprises one or more of thefollowing naturally encoded amino acid substitutions: T79R, K83S, Y85L,T86S, E87S, Q91E (SEQ ID NO: 2 or the corresponding amino acids in SEQID NO: 1 or 3).

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions-37, 45, 46, 89, and 107 (SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NO: 1 or 3). In some embodiments, the hIFNpolypeptide comprises one or more non-naturally encoded amino acids atone or more of the following positions: 37, 45, 46, 89, and 107 (SEQ IDNO: 2 or the corresponding amino acids in SEQ ID NO: 1 or 3) that islinked to a water soluble polymer. In some embodiments, the hIFNpolypeptide comprises one or more non-naturally encoded amino acids atone or more of the following positions: 37, 45, 46, 89, and 107 (SEQ IDNO: 2 or the corresponding amino acids in SEQ ID NO: 1 or 3) that isbonded to a water soluble polymer.

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 37, 45, 46, 89, and 107 and comprises one or more of thefollowing naturally encoded amino acid substitutions: T79R, L80A, Y85L,Y85S, E87S (SEQ ID NO: 2 or the corresponding amino acids in SEQ ID NO:1 or 3). In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 37, 45, 46, 89, and 107 that is linked to a water solublepolymer and comprises one or more of the following naturally encodedamino acid substitutions: T79R, L80A, Y85L, Y85S, E87S (SEQ ID NO: 2 orthe corresponding amino acids in SEQ ID NO: 1 or 3). In someembodiments, the hIFN polypeptide comprises one or more non-naturallyencoded amino acids at one or more of the following positions: 37, 45,46, 89, and 107 that is bonded to a water soluble polymer and comprisesone or more of the following naturally encoded amino acid substitutions:T79R, L80A, Y85L, Y85S, E87S (SEQ ID NO: 2 or the corresponding aminoacids in SEQ ID NO: 1 or 3).

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 23, 24, 25, 26, 27, 28, 30, 31, 32, 33, 128, 129, 131, 132,133, 134, 135, 136, 137, 158, 159, 160, 161, 162, 163, 164, 165 (SEQ IDNO: 2 or the corresponding amino acids in SEQ ID NO: 1 or 3). In someembodiments, the hIFN polypeptide comprises one or more non-naturallyencoded amino acids at one or more of the following positions that islinked or bonded to a water soluble polymer: 23, 24, 25, 26, 27, 28, 30,31, 32, 33, 128, 129, 131, 132, 133, 134, 135, 136, 137, 158, 159, 160,161, 162, 163, 164, 165 (SEQ ID NO: 2 or the corresponding amino acidsin SEQ ID NO: 1 or 3). In some embodiments, the hIFN polypeptidecomprises one or more non-naturally encoded amino acids at one or moreof the following positions: 23, 24, 27, 31, 128, 131, 134, 158 (SEQ IDNO: 2 or the corresponding amino acids in SEQ ID NO: 1 or 3). In someembodiments, the hIFN polypeptide comprises one or more non-naturallyencoded amino acids at one or more of the following positions that islinked or bonded to a water soluble polymer: 23, 24, 27, 31, 128, 131,134, 158 (SEQ ID NO: 2 or the corresponding amino acids in SEQ ID NO: 1or 3). In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 24, 27, 31, 128, 131, 134 (SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NO: 1 or 3). In some embodiments, the hIFNpolypeptide comprises one or more non-naturally encoded amino acids atone or more of the following positions that is linked or bonded to awater soluble polymer: 24, 27, 31, 128, 131, 134 (SEQ ID NO: 2 or thecorresponding amino acids in SEQ ID NO: 1 or 3).

In some embodiments, the IFN polypeptides of the invention comprise oneor more non-naturally encoded amino acids at one or more of thefollowing positions providing an antagonist: 2, 3, 4, 5, 7, 8, 16, 19,20, 40, 42, 50, 51, 58, 68, 69, 70, 71, 73, 97, 105, 109, 112, 118, 148,149, 152, 153, 158, 163, 164, 165, or any combination thereof (SEQ IDNO: 2, or the corresponding amino acids in other IFN's); a hIFNpolypeptide comprising one of these substitutions may potentially act asa weak antagonist or weak agonist depending on the site selected and thedesired activity. Human IFN antagonists include, but are not limited to,those with one or more substitutions at 22, 23, 24, 25, 26, 27, 28, 30,31, 32, 33, 34, 35, 74, 77, 78, 79, 80, 82, 83, 85, 86, 89, 90, 93, 94,124, 125, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137, or anycombination thereof (hIFN; SEQ ID NO: 2 or the corresponding amino acidsin SEQ ID NO: 1 or 3).

In some embodiments, one or more non-naturally encoded amino acids aresubstituted at, but not limited to, one or more of the followingpositions of hIFN (as in SEQ ID NO: 2, or the corresponding amino acidsin other IFN's): 31, 134, 34, 38, 129, 36, 122, 37, 121, 41, 125, 124,149, 117, 39, 118, 120, 107, 108, 106, 100, 111, 113, 114, 41, 45, 46,48, 49, 61, 64, 65, 101, 103, 102, 110, 117, 120, 121, 149, 96, 6, 9,16, 68, 70, 109, 159, 161, 156, 160, 162, 12, 13, 24, 27, 78, 83, 85,87, 89, 164. In one embodiment, a non-naturally encoded amino acid issubstituted at position 38 of hIFN (as in SEQ ID NO: 2, or thecorresponding amino acids in other IFN's). In some embodiments, thenon-naturally occurring amino acid at these or other positions is linkedto a water soluble polymer, including but not limited to positions: 31,134, 34, 38, 129, 36, 122, 37, 121, 41, 125, 124, 149, 117, 39, 118,120, 107, 108, 106, 100, 111, 113, 114, 41, 45, 46, 48, 49, 61, 64, 65,101, 103, 102, 110, 117, 120, 121, 149, 96, 6, 9, 12, 13, 16, 68, 70,109, 159, 161, 156, 160, 162, 24, 27, 78, 83, 85, 87, 89, 164.

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acid and a naturally encoded amino acidsubstitution. In some embodiments, the non-naturally encoded amino acidpresent in the hIFN polypeptide is linked to a water soluble polymer andthe hIFN polypeptide comprises one or more naturally encoded amino acidsubstitution.

A wide variety of non-naturally encoded amino acids can be substitutedfor, or incorporated into, a given position in a hIFN polypeptide. Ingeneral, a particular non-naturally encoded amino acid is selected forincorporation based on an examination of the three dimensional crystalstructure of a hIFN polypeptide with its receptor, a preference forconservative substitutions (i.e., aryl-based non-naturally encoded aminoacids, such as p-acetylphenylalanine or O-propargyltyrosine substitutingfor Phe, Tyr or Trp), and the specific conjugation chemistry that onedesires to introduce into the hIFN polypeptide (e.g., the introductionof 4-azidophenylalanine if one wants to effect a Huisgen[3+2]cycloaddition with a water soluble polymer bearing an alkyne moietyor a amide bond formation with a water soluble polymer that bears anaryl ester that, in turn, incorporates a phosphine moiety).

In one embodiment, the method further includes incorporating into theprotein the unnatural amino acid, where the unnatural amino acidcomprises a first reactive group; and contacting the protein with amolecule (including but not limited to, a label, a dye, a polymer, awater-soluble polymer, a derivative of polyethylene glycol, aphotocrosslinker, a radionuclide, a cytotoxic compound, a drug, anaffinity label, a photoaffinity label, a reactive compound, a resin, asecond protein or polypeptide or polypeptide analog, an antibody orantibody fragment, a metal chelator, a cofactor, a fatty acid, acarbohydrate, a polynucleotide, a DNA, a RNA, an antisensepolynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin,an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spinlabel, a fluorophore, a metal-containing moiety, a radioactive moiety, anovel functional group, a group that covalently or noncovalentlyinteracts with other molecules, a photocaged moiety, an actinicradiation excitable moiety, a photoisomerizable moiety, biotin, aderivative of biotin, a biotin analogue, a moiety incorporating a heavyatom, a chemically cleavable group, a photocleavable group, an elongatedside chain, a carbon-linked sugar, a redox-active agent, an aminothioacid, a toxic moiety, an isotopically labeled moiety, a biophysicalprobe, a phosphorescent group, a chemiluminescent group, an electrondense group, a magnetic group, an intercalating group, a chromophore, anenergy transfer agent, a biologically active agent, a detectable label,a small molecule, a quantum dot, a nanotransmitter, a radionucleotide, aradiotransmitter, a neutron-capture agent, or any combination of theabove, or any other desirable compound or substance) that comprises asecond reactive group. The first reactive group reacts with the secondreactive group to attach the molecule to the unnatural amino acidthrough a [3+2]cycloaddition. In one embodiment, the first reactivegroup is an alkynyl or azido moiety and the second reactive group is anazido or alkynyl moiety. For example, the first reactive group is thealkynyl moiety (including but not limited to, in unnatural amino acidp-propargyloxyphenylalanine) and the second reactive group is the azidomoiety. In another example, the first reactive group is the azido moiety(including but not limited to, in the unnatural amino acidp-azido-L-phenylalanine) and the second reactive group is the alkynylmoiety.

In some cases, the non-naturally encoded amino acid substitution(s) willbe combined with other additions, substitutions or deletions within thehIFN polypeptide to affect other biological traits of the hIFNpolypeptide. In some cases, the other additions, substitutions ordeletions may increase the stability (including but not limited to,resistance to proteolytic degradation) of the hIFN polypeptide orincrease affinity of the hIFN polypeptide for its receptor. In somecases, the other additions, substitutions or deletions may increase thesolubility (including but not limited to, when expressed in E. coli orother host cells) of the hIFN polypeptide. In some embodimentsadditions, substitutions or deletions may increase the polypeptidesolubility following expression in E. coli or other recombinant hostcells. In some embodiments sites are selected for substitution with anaturally encoded or non-natural amino acid in addition to another sitefor incorporation of a non-natural amino acid that results in increasingthe polypeptide solubility following expression in E. coli recombinanthost cells. In some embodiments, the hIFN polypeptides comprise anotheraddition, substitution or deletion that modulates affinity for the hIFNpolypeptide receptor, modulates (including but not limited to, increasesor decreases) receptor dimerization, modulated downstream signalingevents after hIFN receptor binding, stabilizes receptor dimers,modulates circulating half-life, modulates release or bio-availability,facilitates purification, or improves or alters a particular route ofadministration. hIFN polypeptides of the invention may modulate one ormore of the biological activities of IFN including side effects foundwith current IFN therapeutics. hIFN polypeptides of the invention maymodulate toxicity found with current IFN therapeutics. Similarly, hIFNpolypeptides can comprise chemical or enzyme cleavage sequences,protease cleavage sequences, reactive groups, antibody-binding domains(including but not limited to, FLAG or poly-His) or other affinity basedsequences (including, but not limited to, FLAG, poly-His, GST, etc.) orlinked molecules (including, but not limited to, biotin) that improvedetection (including, but not limited to, GFP), purification, transportthrough tissues or membranes, prodrug release or activation, hIFN sizereduction, or other traits of the polypeptide.

In some embodiments, the substitution of a non-naturally encoded aminoacid generates a hIFN antagonist. A subset of exemplary sites forincorporation of one or more non-naturally encoded amino acid include:2, 3, 4, 5, 7, 8, 16, 19, 20, 40, 42, 50, 51, 58, 68, 69, 70, 71, 73,97, 105, 109, 112, 118, 148, 149, 152, 153, 158, 163, 164, 165 (as inSEQ ID NO: 2, or the corresponding amino acids in other IFNs). Anothersubset of exemplary sites for incorporation of a non-naturally encodedamino acid include: 22, 23, 24, 25, 26, 27, 28, 30, 31, 32, 33, 34, 35,74, 77, 78, 79, 80, 82, 83, 85, 86, 89, 90, 93, 94, 124, 125, 127, 128,129, 131, 132, 133, 134, 135, 136, 137, (hIFN; SEQ ID NO: 2 or thecorresponding amino acids in SEQ ID NO: 1 or 3). In some embodiments,hIFN antagonists comprise at least one substitution in the regions 1-9(N-terminus), 10-21 (A helix), 22-39 (region between A helix and Bhelix), 40-75 (B helix), 76-77 (region between B helix and C helix),78-100 (C helix), 101-110 (region between C helix and D helix), 111-132(D helix), 133-136 (region between D and E helix), 137-155 (E helix),156-165 (C-terminus) that cause IFN to act as an antagonist. In otherembodiments, the exemplary sites of incorporation of a non-naturallyencoded amino acid include residues within the amino terminal region ofhelix A and a portion of helix C. In other embodiments, the above-listedsubstitutions are combined with additional substitutions that cause thehIFN polypeptide to be a hIFN antagonist. In some embodiments, the hIFNantagonist comprises a non-naturally encoded amino acid linked to awater soluble polymer that is present in a receptor binding region ofthe hIFN molecule.

In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids aresubstituted with one or more non-naturally-encoded amino acids. In somecases, the hIFN polypeptide further includes 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more substitutions of one or more non-naturally encoded aminoacids for naturally-occurring amino acids. In some embodiments, one ormore residues in the following regions of hIFN are substituted with oneor more non-naturally encoded amino acids: 1-9 (N-terminus), 10-21 (Ahelix), 22-39 (region between A helix and B helix), 40-75 (B helix),76-77 (region between B helix and C helix), 78-100 (C helix), 101-110(region between C helix and D helix), 111-132 (D helix), 133-136 (regionbetween D and E helix), 137-155 (E helix), 156-165 (C-terminus). In somecases, one or more non-naturally encoded residues are linked to one ormore lower molecular weight linear or branched PEGs (approximately 5-20kDa in mass or less), thereby enhancing binding affinity and comparableserum half-life relative to the species attached to a single, highermolecular weight PEG.

Sites for incorporation in hIFN of one or more non-naturally encodedamino acids include combinations of the following residues (as in SEQ IDNO: 2, or the corresponding amino acids in other IFN's orinterferon-like cytokines): before position 1 (i.e. at the N-terminus),1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 13, 16, 19, 20, 22, 23, 24, 25, 26, 27,28, 30, 31, 32, 33, 34, 35, 40, 41, 42, 45, 46, 48, 49, 50, 51, 58, 61,64, 65, 68, 69, 70, 71, 73, 74, 77, 78, 79, 80, 81, 82, 83, 85, 86, 89,90, 93, 94, 96, 97, 100, 101, 103, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 117, 118, 120, 121, 124, 125, 127, 128, 129, 131, 132,133, 134, 135, 136, 137, 148, 149, 152, 153, 156, 158, 159, 160, 161,162, 163, 164, 165, 166 (i.e. at the carboxyl terminus of the protein)or any combination thereof. Sites for incorporation in hIFN of one ormore non-naturally encoded amino acids include combinations of thefollowing residues (as in SEQ ID NO: 2, or the corresponding amino acidsin other IFN's or interferon-like cytokines): 31, 134, 34, 38, 129, 36,122, 37, 121, 41, 125, 124, 149, 117, 39, 118, 120, 107, 108, 106, 100,111, 113, 114, 41, 45, 46, 48, 49, 61, 64, 65, 101, 103, 102, 110, 117,120, 121, 149, 96, 6, 9, 16, 68, 70, 109, 159, 161, 156, 160, 162, 12,13, 24, 27, 78, 83, 85, 87, 89, 164.

VII. Expression in Non-Eukaryotes and Eukaryotes

To obtain high level expression of a cloned hIFN polynucleotide, onetypically subclones polynucleotides encoding a hIFN polypeptide of theinvention into an expression vector that contains a strong promoter todirect transcription, a transcription/translation terminator, and if fora nucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are known tothose of ordinary skill in the art and described, e.g., in Sambrook etal. and Ausubel et al.

Bacterial expression systems for expressing hIFN polypeptides of theinvention are available in, including but not limited to, E. coli,Bacillus sp., Pseudomonas fluorescens, Pseudomonas aeruginosa,Pseudomonas putida, and Salmonella (Palva et al., Gene 22:229-235(1983); Mosbach et al., Nature 302:543-545 (1983)). Kits for suchexpression systems are commercially available. Eukaryotic expressionsystems for mammalian cells, yeast, and insect cells are known to thoseof ordinary skill in the art and are also commercially available. Incases where orthogonal tRNAs and aminoacyl tRNA synthetases (describedabove) are used to express the hIFN polypeptides of the invention, hostcells for expression are selected based on their ability to use theorthogonal components. Exemplary host cells include Gram-positivebacteria (including but not limited to B. brevis, B. subtilis, orStreptomyces) and Gram-negative bacteria (E. coli, Pseudomonasfluorescens, Pseudomonas aeruginosa, Pseudomonas putida), as well asyeast and other eukaryotic cells. Cells comprising O-tRNA/O-RS pairs canbe used as described herein.

A eukaryotic host cell or non-eukaryotic host cell of the presentinvention provides the ability to synthesize proteins that compriseunnatural amino acids in large useful quantities. In one aspect, thecomposition optionally includes, including but not limited to, at least10 micrograms, at least 50 micrograms, at least 75 micrograms, at least100 micrograms, at least 200 micrograms, at least 250 micrograms, atleast 500 micrograms, at least 1 milligram, at least 10 milligrams, atleast 100 milligrams, at least one gram, or more of the protein thatcomprises an unnatural amino acid, or an amount that can be achievedwith in vivo protein production methods (details on recombinant proteinproduction and purification are provided herein). In another aspect, theprotein is optionally present in the composition at a concentration of,including but not limited to, at least 10 micrograms of protein perliter, at least 50 micrograms of protein per liter, at least 75micrograms of protein per liter, at least 100 micrograms of protein perliter, at least 200 micrograms of protein per liter, at least 250micrograms of protein per liter, at least 500 micrograms of protein perliter, at least 1 milligram of protein per liter, or at least 10milligrams of protein per liter or more, in, including but not limitedto, a cell lysate, a buffer, a pharmaceutical buffer, or other liquidsuspension (including but not limited to, in a volume of, including butnot limited to, anywhere from about 1 ni to about 100 L or more). Theproduction of large quantities (including but not limited to, greaterthat that typically possible with other methods, including but notlimited to, in vitro translation) of a protein in a eukaryotic cellincluding at least one unnatural amino acid is a feature of theinvention.

A eukaryotic host cell or non-eukaryotic host cell of the presentinvention provides the ability to biosynthesize proteins that compriseunnatural amino acids in large useful quantities. For example, proteinscomprising an unnatural amino acid can be produced at a concentrationof, including but not limited to, at least 10 μg/liter, at least 50μg/liter, at least 75 μg/liter, at least 100 μg/liter, at least 200μg/liter, at least 250 μg/liter, or at least 500 μg/liter, at least 1mg/liter, at least 2 mg/liter, at least 3 mg/liter, at least 4 mg/liter,at least 5 mg/liter, at least 6 mg/liter, at least 7 mg/liter, at least8 mg/liter, at least 9 mg/liter, at least 10 mg/liter, at least 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900mg/liter, 1 g/liter, 5 g/liter, 10 g/liter or more of protein in a cellextract, cell lysate, culture medium, a buffer, and/or the like.

I. Expression Systems, Culture, and Isolation

hIFN polypeptides may be expressed in any number of suitable expressionsystems including, for example, yeast, insect cells, mammalian cells,and bacteria. A description of exemplary expression systems is providedbelow.

Yeast As used herein, the term “yeast” includes any of the variousyeasts capable of expressing a gene encoding a hIFN polypeptide. Suchyeasts include, but are not limited to, ascosporogenous yeasts(Endomycetales), basidiosporogenous yeasts and yeasts belonging to theFungi imperfecti (Blastomycetes) group. The ascosporogenous yeasts aredivided into two families, Spermophthoraceae and Saccharomycelaceae. Thelatter is comprised of four subfamilies, Schizosaccharomycoideae (e.g.,genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae andSaccharomycoideae (e.g., genera Pichia, Kluyveromyces andSaccharomyces). The basidiosporogenous yeasts include the generaLeucosporiditim, Rhodosporidium, Sporidiobolus, Filobasidium, andFilobasidiella. Yeasts belonging to the Fungi Imperfecti (Blastomycetes)group are divided into two families, Sporobolomycetaceae (e.g., generaSporobolomyces and Bullera) and Cryptococcaceae (e.g., genus Candida).

Of particular interest for use with the present invention are specieswithin the genera Pichia, Kluyveromyces, Saccharomyces,Schizosaccharomyces, Hansenula, Torulopsis, and Candida, including, butnot limited to, P. pastoris, P. guillerimondii, & cerevisiae, S.carlsbergensis, S. diastaticus, S. douglasii, S. kluyveri, S. norbensis,S. oviformis, K. lactis, K. fragilis, C. albicans, C. maltosa, and H.polymorpha.

The selection of suitable yeast for expression of hIFN polypeptides iswithin the skill of one of ordinary skill in the art. In selecting yeasthosts for expression, suitable hosts may include those shown to have,for example, good secretion capacity, low proteolytic activity, goodsecretion capacity, good soluble protein production, and overallrobustness. Yeast are generally available from a variety of sourcesincluding, but not limited to, the Yeast Genetic Stock Center,Department of Biophysics and Medical Physics, University of California(Berkeley, Calif.), and the American Type Culture Collection (“ATCC”)(Manassas, Va.).

The term “yeast host” or “yeast host cell” includes yeast that can be,or has been, used as a recipient for recombinant vectors or othertransfer DNA. The term includes the progeny of the original yeast hostcell that has received the recombinant vectors or other transfer DNA. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell that are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding a hIFN polypeptide, areincluded in the progeny intended by this definition.

Expression and transformation vectors, including extrachromosomalreplicons or integrating vectors, have been developed for transformationinto many yeast hosts. For example, expression vectors have beendeveloped for S. cerevisiae (Sikorski et al., GENETICS (1989) 122:19;Ito et al., J. BACTERIOL. (1983) 153:163; Hinnen et al., PROC. NATL.ACAD. SCI. USA (1978) 75:1929); C. albicans (Kurtz et al., MOL. CELL.BIOL. (1986) 6:142); C. maltosa (Kunze et al., J. BASIC MICROBIOL*.(1985) 25:141); H. polymorpha (Gleeson et al., J. GEN. MICROBIOL. (1986)132:3459; Roggenkamp et al., MOL. GENETICS AND GENOMICS (1986) 202:302);K. fragilis (Das et al., J. BACTERIOL. (1984) 158:1165); K. lactis (DeLouvencourt et al., J. BACTERIOL. (1983) 154:737; Van den Berg et al.,BIOTECHNOLOGY (NY) (1990) 8:135); P. guillerimondii (Kunze et al., J.BASIC MICROBIOL. (1985) 25:141); P. pastoris (U.S. Pat. Nos. 5,324,639;4,929,555; and 4,837,148; Cregg et al., MOL. CELL. BIOL. (1985) 5:3376);Schizosaccharomyces pombe (Beach et al., NATURE (1982) 300:706); and Y.lipolytica; A. nidulans (Ballance et al., BIOCHEM. BIOPHYS. RES. COMMUN.(1983) 112:284-89; Tilburn et al., GENE (1983) 26:205-221; and Yelton etal., PROC. NATL. ACAD. SCI. USA (1984) 81:1470-74); A. niger (Kelly andHynes, EMBO J. (1985) 4:475-479); T. reesia (EP 0 244 234); andfilamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium(WO 91/00357), each incorporated by reference herein.

Control sequences for yeast vectors are well known to those of ordinaryskill in the art and include, but are not limited to, promoter regionsfrom genes such as alcohol dehydrogenase (ADH) (EP 0 284 044); enolase;glucokinase; glucose-6-phosphate isomerase;glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH); hexokinase;phosphofructokinase; 3-phosphoglycerate mutase; and pyruvate kinase(PyK) (EP 0 329 203). The yeast PHO5 gene, encoding acid phosphatase,also may provide useful promoter sequences (Miyanohara et al., PROC.NATL. ACAD. SCI. USA (1983) 80:1). Other suitable promoter sequences foruse with yeast hosts may include the promoters for 3-phosphoglyceratekinase (Hitzeman et al., J. BIOL. CHEM. (1980) 255:12073); and otherglycolytic enzymes, such as pyruvate decarboxylase, triosephosphateisomerase, and phosphoglucose isomerase (Holland et al., BIOCHEMISTRY(1978) 17:4900; Hess et al., J. ADV. ENZYME REG. (1969) 7:149).Inducible yeast promoters having the additional advantage oftranscription controlled by growth conditions may include the promoterregions for alcohol dehydrogenase 2; isocytochrome C; acid phosphatase;metallothionein; glyceraldehyde-3-phosphate dehydrogenase; degradativeenzymes associated with nitrogen metabolism; and enzymes responsible formaltose and galactose utilization. Suitable vectors and promoters foruse in yeast expression are further described in EP 0 073 657.

Yeast enhancers also may be used with yeast promoters. In addition,synthetic promoters may also function as yeast promoters. For example,the upstream activating sequences (UAS) of a yeast promoter may bejoined with the transcription activation region of another yeastpromoter, creating a synthetic hybrid promoter. Examples of such hybridpromoters include the ADH regulatory sequence linked to the GAPtranscription activation region. See U.S. Pat. Nos. 4,880,734 and4,876,197, which are incorporated by reference herein. Other examples ofhybrid promoters include promoters that consist of the regulatorysequences of the ADH2, GAL4, GAL10, or PHO5 genes, combined with thetranscriptional activation region of a glycolytic enzyme gene such asGAP or PyK. See EP 0 164 556. Furthermore, a yeast promoter may includenaturally occurring promoters of non-yeast origin that have the abilityto bind yeast RNA polymerase and initiate transcription.

Other control elements that may comprise part of the yeast expressionvectors include terminators, for example, from GAPDH or the enolasegenes (Holland et al., J. BIOL. CHEM. (1981) 256:1385). In addition, theorigin of replication from the 2μ plasmid origin is suitable for yeast.A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid. See Tschumper et al., GENE (1980) 10:157; Kingsman etal., GENE (1979) 7:141. The trp1 gene provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan.Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

Methods of introducing exogenous DNA into yeast hosts are known to thoseof ordinary skill in the art, and typically include, but are not limitedto, either the transformation of spheroplasts or of intact yeast hostcells treated with alkali cations. For example, transformation of yeastcan be carried out according to the method described in Hsiao et al.,PROC. NATL. ACAD. SCI. USA (1979) 76:3829 and Van Solingen et al., J.BACT. (1977) 130:946. However, other methods for introducing DNA intocells such as by nuclear injection, electroporation, or protoplastfusion may also be used as described generally in SAMBROOK ET AL.,MOLECULAR CLONING: A LAB. MANUAL (2001). Yeast host cells may then becultured using standard techniques known to those of ordinary skill inthe art.

Other methods for expressing heterologous proteins in yeast host cellsare known to those of ordinary skill in the art. See generally U.S.Patent Publication No. 20020055169, U.S. Pat. Nos. 6,361,969; 6,312,923;6,183,985; 6,083,723; 6,017,731; 5,674,706; 5,629,203; 5,602,034; and5,089,398; U.S. Reexamined Pat. Nos. RE37,343 and RE35,749; PCTPublished Patent Applications WO 99/07862; WO 98/37208; and WO 98/26080;European Patent Applications EP 0 946 736; EP 0 732 403; EP 0 480 480;WO 90/10277; EP 0 340 986; EP 0 329 203; EP 0 324 274; and EP 0 164 556.See also Gellissen et al., ANTONIE VAN LEEUWENHOEK (1992) 62(1-2):79-93;Romanos et al., YEAST (1992) 8(6):423-488; Goeddel, METHODS INENZYMOLOGY (1990) 185:3-7, each incorporated by reference herein.

The yeast host strains may be grown in fermentors during theamplification stage using standard feed batch fermentation methods knownto those of ordinary skill in the art. The fermentation methods may beadapted to account for differences in a particular yeast host's carbonutilization pathway or mode of expression control. For example,fermentation of a Saccharomyces yeast host may require a single glucosefeed, complex nitrogen source (e.g., casein hydrolysates), and multiplevitamin supplementation. In contrast, the methylotrophic yeast P.pastoris may require glycerol, methanol, and trace mineral feeds, butonly simple ammonium (nitrogen) salts for optimal growth and expression.See, e.g., U.S. Pat. No. 5,324,639; Elliott et al., J. PROTEIN CHEM.(1990) 9:95; and Fieschko et al., BIOTECH. BIOENG. (1987) 29:1113,incorporated by reference herein.

Such fermentation methods, however, may have certain common featuresindependent of the yeast host strain employed. For example, a growthlimiting nutrient, typically carbon, may be added to the fermentorduring the amplification phase to allow maximal growth. In addition,fermentation methods generally employ a fermentation medium designed tocontain adequate amounts of carbon, nitrogen, basal salts, phosphorus,and other minor nutrients (vitamins, trace minerals and salts, etc.).Examples of fermentation media suitable for use with Pichia aredescribed in U.S. Pat. Nos. 5,324,639 and 5,231,178, which areincorporated by reference herein.

Baculovirus-Infected Insect Cells The term “insect host” or “insect hostcell” refers to a insect that can be, or has been, used as a recipientfor recombinant vectors or other transfer DNA. The term includes theprogeny of the original insect host cell that has been transfected. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell that are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding a hIFN polypeptide, areincluded in the progeny intended by this definition.

The selection of suitable insect cells for expression of hIFNpolypeptides is known to those of ordinary skill in the art. Severalinsect species are well described in the art and are commerciallyavailable including Aedes aegypti, Bombyx mori, Drosophila melanogaster,Spodoptera frugiperda, and Trichoplusia ni. In selecting insect hostsfor expression, suitable hosts may include those shown to have, interalia, good secretion capacity, low proteolytic activity, and overallrobustness. Insect are generally available from a variety of sourcesincluding, but not limited to, the Insect Genetic Stock Center,Department of Biophysics and Medical Physics, University of California(Berkeley, Calif.); and the American Type Culture Collection (“ATCC”)(Manassas, Va.).

Generally, the components of a baculovirus-infected insect expressionsystem include a transfer vector, usually a bacterial plasmid, whichcontains both a fragment of the baculovirus genome, and a convenientrestriction site for insertion of the heterologous gene to be expressed;a wild type baculovirus with sequences homologous to thebaculovirus-specific fragment in the transfer vector (this allows forthe homologous recombination of the heterologous gene in to thebaculovirus genome); and appropriate insect host cells and growth media.The materials, methods and techniques used in constructing vectors,transfecting cells, picking plaques, growing cells in culture, and thelike are known in the art and manuals are available describing thesetechniques.

After inserting the heterologous gene into the transfer vector, thevector and the wild type viral genome are transfected into an insecthost cell where the vector and viral genome recombine. The packagedrecombinant virus is expressed and recombinant plaques are identifiedand purified. Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, forexample, Invitrogen Corp. (Carlsbad, Calif.). These techniques aregenerally known to those of ordinary skill in the art and fullydescribed in SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT STATIONBULLETIN NO. 1555 (1987), herein incorporated by reference. See also,RICHARDSON, 39 METHODS IN MOLECULAR BIOLOGY: BACULOVIRUS EXPRESSIONPROTOCOLS (1995); AUSUBEL ET AL., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY16.9-16.11 (1994); KING AND POSSEE, THE BACULOVIRUS SYSTEM: A LABORATORYGUIDE (1992); and O'REILLY ET AL., BACULOVIRUS EXPRESSION VECTORS: ALABORATORY MANUAL (1992).

Indeed, the production of various heterologous proteins usingbaculovirus/insect cell expression systems is known to those of ordinaryskill in the art. See, e.g., U.S. Pat. Nos. 6,368,825; 6,342,216;6,338,846; 6,261,805; 6,245,528, 6,225,060; 6,183,987; 6,168,932;6,126,944; 6,096,304; 6,013,433; 5,965,393; 5,939,285; 5,891,676;5,871,986; 5,861,279; 5,858,368; 5,843,733; 5,762,939; 5,753,220;5,605,827; 5,583,023; 5,571,709; 5,516,657; 5,290,686; WO 02/06305; WO01/90390; WO 01/27301; WO 01/05956; WO 00/55345; WO 00/20032; WO99/51721; WO 99/45130; WO 99/31257; WO 99/10515; WO 99/09193; WO97/26332; WO 96/29400; WO 96/25496; WO 96/06161; WO 95/20672; WO93/03173; WO 92/16619; WO 92/02628; WO 92/01801; WO 90/14428; WO90/10078; WO 90/02566; WO 90/02186; WO 90/01556; WO 89/01038; WO89/01037; WO 88/07082, which are incorporated by reference herein.

Vectors that are useful in baculovirus/insect cell expression systemsare known in the art and include, for example, insect expression andtransfer vectors derived from the baculovirus Autographacalifornicanuclear polyhedrosis virus (AcNPV), which is a helper-independent, viralexpression vector. Viral expression vectors derived from this systemusually use the strong viral polyhedrin gene promoter to driveexpression of heterologous genes. See generally, O'Reilly ET AL.,BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992).

Prior to inserting the foreign gene into the baculovirus genome, theabove-described components, comprising a promoter, leader (if desired),coding sequence of interest, and transcription termination sequence, aretypically assembled into an intermediate transplacement construct(transfer vector). Intermediate transplacement constructs are oftenmaintained in a replicon, such as an extra chromosomal element (e.g.,plasmids) capable of stable maintenance in a host, such as bacteria. Thereplicon will have a replication system, thus allowing it to bemaintained in a suitable host for cloning and amplification. Morespecifically, the plasmid may contain the polyhedrin polyadenylationsignal (Miller, ANN. REV. MICROBIOL. (1988) 42:177) and a prokaryoticampicillin-resistance (amp) gene and origin of replication for selectionand propagation in E. coli.

One commonly used transfer vector for introducing foreign genes intoAcNPV is pAc373. Many other vectors, known to those of skill in the art,have also been designed including, for example, pVL985, which alters thepolyhedrin start codon from ATG to ATT, and which introduces a BamHIcloning site 32 base pairs downstream from the ATT. See Luckow andSummers, VIROLOGY 170:31 (1989). Other commercially available vectorsinclude, for example, PBlueBac4.5/V5-His; pBlueBacHis2; pMelBac;pBlueBac4.5 (Invitrogen Corp., Carlsbad, Calif.).

After insertion of the heterologous gene, the transfer vector and wildtype baculoviral genome are co-transfected into an insect cell host.Methods for introducing heterologous DNA into the desired site in thebaculovirus virus are known in the art. See SUMMERS AND SMITH, TEXASAGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987); Smith et al.,MOL. CELL. BIOL. (1983) 3:2156; Luckow and Summers, VIROLOGY (1989)170:31. For example, the insertion can be into a gene such as thepolyhedrin gene, by homologous double crossover recombination; insertioncan also be into a restriction enzyme site engineered into the desiredbaculovirus gene. See Miller et al., BIOESSAYS (1989) 11(4):91.

Transfection may be accomplished by electroporation. See TROTTER ANDWOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Mann and King, J. GEN.VIROL. (1989) 70:3501. Alternatively, liposomes may be used to transfectthe insect cells with the recombinant expression vector and thebaculovirus. See, e.g., Liebman et al., BIOTECHNIQUES (1999) 26(1):36;Graves et al., BIOCHEMISTRY (1998) 37:6050; Nomura et al., J. BIOL.CHEM. (1998) 273(22):13570; Schmidt et al., PROTEIN EXPRESSION ANDPURIFICATION (1998) 12:323; Siffert et al., NATURE GENETICS (1998)18:45; TILKINS ET AL., CELL BIOLOGY: A LABORATORY HANDBOOK 145-154(1998); Cai et al., PROTEIN EXPRESSION AND PURIFICATION (1997) 10:263;Dolphin et al., NATURE GENETICS (1997) 17:491; Kost et al., GENE (1997)190:139; Jakobsson et al., J. BIOL. CHEM. (1996) 271:22203; Rowles etal., J. BIOL. CHEM. (1996) 271(37):22376; Reverey et al., J. BIOL. CHEM.(1996) 271(39):23607-10; Stanley et al., J. BIOL. CHEM. (1995) 270:4121;Sisk et al., J. VIROL. (1994) 68(2):766; and Peng et al., BIOTECHNIQUES(1993) 14(2):274. Commercially available liposomes include, for example,Cellfectin® and Lipofectin® (Invitrogen, Corp., Carlsbad, Calif.). Inaddition, calcium phosphate transfection may be used. See TROTTER ANDWOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Kitts, NAR (1990)18(19):5667; and Mann and King, J. GEN. VIROL. (1989) 70:3501.

Baculovirus expression vectors usually contain a baculovirus promoter. Abaculovirus promoter is any DNA sequence capable of binding abaculovirus RNA polymerase and initiating the downstream (3′)transcription of a coding sequence (e.g., structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region typically includes an RNA polymerase binding site anda transcription initiation site. A baculovirus promoter may also have asecond domain called an enhancer, which, if present, is usually distalto the structural gene. Moreover, expression may be either regulated orconstitutive.

Structural genes, abundantly transcribed at late times in the infectioncycle, provide particularly useful promoter sequences. Examples includesequences derived from the gene encoding the viral polyhedron protein(FRIESEN ET AL ., The Regulation of Baculovirus Gene Expression in THEMOLECULAR BIOLOGY OF BACULOVIRUSES (1986); EP 0 127 839 and 0 155 476)and the gene encoding the p10 protein (Vlak et al., J. GEN. VIROL.(1988) 69:765).

The newly formed baculovirus expression vector is packaged into aninfectious recombinant baculovirus and subsequently grown plaques may bepurified by techniques known to those of ordinary skill in the art. SeeMiller et al., BIOESSAYS (1989) 11(4):91; SUMMERS AND SMITH, TEXASAGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987).

Recombinant baculovirus expression vectors have been developed forinfection into several insect cells. For example, recombinantbaculoviruses have been developed for, inter alia, Aedes aegypti (ATCCNo. CCL-125), Bombyx mori (ATCC No. CRL-8910), Drosophila melanogaster(ATCC No. 1963), Spodoptera frugiperda, and Trichoplusia ni. See Wright,NATURE (1986) 321:718; Carbonell et al., J. VIROL. (1985) 56:153; Smithet al., MOL. CELL. BIOL. (1983) 3:2156. See generally, Fraser et al., INVITRO CELL. DEV. BIOL. (1989) 25:225. More specifically, the cell linesused for baculovirus expression vector systems commonly include, but arenot limited to, Sf9 (Spodoptera frugiperda) (ATCC No. CRL-1711), Sf21(Spodoptera frugiperda) (Invitrogen Corp., Cat. No. 11497-013 (Carlsbad,Calif.)), Tri-368 (Trichopulsia ni), and High-Five™ BTI-TN-5B1-4(Trichopulsia ni).

Cells and culture media are commercially available for both direct andfusion expression of heterologous polypeptides in abaculovirus/expression, and cell culture technology is generally knownto those of ordinary skill in the art.

E. Coli, Pseudomonas species, and other Prokaryotes Bacterial expressiontechniques are known to those of ordinary skill in the art. A widevariety of vectors are available for use in bacterial hosts. The vectorsmay be single copy or low or high multicopy vectors. Vectors may servefor cloning and/or expression. In view of the ample literatureconcerning vectors, commercial availability of many vectors, and evenmanuals describing vectors and their restriction maps andcharacteristics, no extensive discussion is required here. As iswell-known, the vectors normally involve markers allowing for selection,which markers may provide for cytotoxic agent resistance, prototrophy orimmunity. Frequently, a plurality of markers is present, which providefor different characteristics.

A bacterial promoter is any DNA sequence capable of binding bacterialRNA polymerase and initiating the downstream (3′) transcription of acoding sequence (e.g. structural gene) into mRNA. A promoter will have atranscription initiation region which is usually placed proximal to the5′ end of the coding sequence. This transcription initiation regiontypically includes an RNA polymerase binding site and a transcriptioninitiation site. A bacterial promoter may also have a second domaincalled an operator, that may overlap an adjacent RNA polymerase bindingsite at which RNA synthesis begins. The operator permits negativeregulated (inducible) transcription, as a gene repressor protein maybind the operator and thereby inhibit transcription of a specific gene.Constitutive expression may occur in the absence of negative regulatoryelements, such as the operator. In addition, positive regulation may beachieved by a gene activator protein binding sequence, which, if presentis usually proximal (5′) to the RNA polymerase binding sequence. Anexample of a gene activator protein is the catabolite activator protein(CAP), which helps initiate transcription of the lac operon inEscherichia coli (E. coli) [Raibaud et al., ANNU. REV. GENET. (1984)18:173]. Regulated expression may therefore be either positive ornegative, thereby either enhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) [Chang etal., NATURE (1977) 198:1056], and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp) [Goeddel et al., NUC. ACIDS RES. (1980) 8:4057; Yelverton et al.,NUCL. ACIDS RES. (1981) 9:731; U.S. Pat. No. 4,738,921; EP Pub. Nos. 036776 and 121 775, which are incorporated by reference herein]. Theβ-galactosidase (bla) promoter system [Weissmann (1981) “The cloning ofinterferon and other mistakes,” In Interferon 3 (Ed. I. Gresser)],bacteriophage lambda PL [Shimatake et al., NATURE (1981) 292:128] and T5[U.S. Pat. No. 4,689,406, which are incorporated by reference herein]promoter systems also provide useful promoter sequences. Preferredmethods of the present invention utilize strong promoters, such as theT7 promoter to induce hIFN polypeptides at high levels. Examples of suchvectors are known to those of ordinary skill in the art and include thepET29 series from Novagen, and the pPOP vectors described in WO99/05297,which is incorporated by reference herein. Such expression systemsproduce high levels of hIFN polypeptides in the host withoutcompromising host cell viability or growth parameters. pET19 (Novagen)is another vector known in the art.

In addition, synthetic promoters which do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of one bacterial or bacteriophage promoter may be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433, which isincorporated by reference herein]. For example, the tac promoter is ahybrid trp-lac promoter comprised of both trp promoter and lac operonsequences that is regulated by the lac repressor [Amann et al., GENE(1983) 25:167; de Boer et al., PROC. NATL. ACAD. SCI. (1983) 80:21].Furthermore, a bacterial promoter can include naturally occurringpromoters of non-bacterial origin that have the ability to bindbacterial RNA polymerase and initiate transcription. A naturallyoccurring promoter of non-bacterial origin can also be coupled with acompatible RNA polymerase to produce high levels of expression of somegenes in prokaryotes. The bacteriophage T7 RNA polymerase/promotersystem is an example of a coupled promoter system [Studier et al., J.MOL. BIOL. (1986) 189:113; Tabor et al., Proc Natl. Acad. Sci. (1985)82:1074]. In addition, a hybrid promoter can also be comprised of abacteriophage promoter and an E. coli operator region (EP Pub. No. 267851).

In addition to a functioning promoter sequence, an efficient ribosomebinding site is also useful for the expression of foreign genes inprokaryotes. In E. coli, the ribosome binding site is called theShine-Dalgarno (SD) sequence and includes an initiation codon (ATG) anda sequence 3-9 nucleotides in length located 3-11 nucleotides upstreamof the initiation codon [Shine et al., NATURE (1975) 254:34]. The SDsequence is thought to promote binding of mRNA to the ribosome by thepairing of bases between the SD sequence and the 3′ and of E. coli 16SrRNA [Steitz et al. “Genetic signals and nucleotide sequences inmessenger RNA”, In Biological Regulation and Development: GeneExpression (Ed. R. F. Goldberger, 1979)]. To express eukaryotic genesand prokaryotic genes with weak ribosome-binding site [Sambrook et al.“Expression of cloned genes in Escherichia coli”, Molecular Cloning: ALaboratory Manual, 1989].

The term “bacterial host” or “bacterial host cell” refers to a bacterialthat can be, or has been, used as a recipient for recombinant vectors orother transfer DNA. The term includes the progeny of the originalbacterial host cell that has been transfected. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement to theoriginal parent, due to accidental or deliberate mutation. Progeny ofthe parental cell that are sufficiently similar to the parent to becharacterized by the relevant property, such as the presence of anucleotide sequence encoding a hIFN polypeptide, are included in theprogeny intended by this definition.

The selection of suitable host bacteria for expression of hIFNpolypeptides is known to those of ordinary skill in the art. Inselecting bacterial hosts for expression, suitable hosts may includethose shown to have, inter alia, good inclusion body formation capacity,low proteolytic activity, and overall robustness. Bacterial hosts aregenerally available from a variety of sources including, but not limitedto, the Bacterial Genetic Stock Center, Department of Biophysics andMedical Physics, University of California (Berkeley, Calif.); and theAmerican Type Culture Collection (“ATCC”) (Manassas, Va.).Industrial/pharmaceutical fermentation generally use bacterial derivedfrom K strains (e.g. W3110) or from bacteria derived from B strains(e.g. BL21). These strains are particularly useful because their growthparameters are extremely well known and robust. In addition, thesestrains are non-pathogenic, which is commercially important for safetyand environmental reasons. Other examples of suitable E. coli hostsinclude, but are not limited to, strains of BL21, DH10B, or derivativesthereof. In another embodiment of the methods of the present invention,the E. coli host is a protease minus strain including, but not limitedto, OMP- and LON-. The host cell strain may be a species of Pseudomonas,including but not limited to, Pseudomonas fluorescens, Pseudomonasaeruginosa, and Pseudomonas putida. Pseudomonas fluorescens biovar 1,designated strain MB101, is known to be useful for recombinantproduction and is available for therapeutic protein productionprocesses. Examples of a Pseudomonas expression system include thesystem available from The Dow Chemical Company as a host strain(Midland, Mich. available on the World Wide Web at dow.com). U.S. Pat.Nos. 4,755,465 and 4,859,600, which are incorporated by referenceherein, describe the use of Pseudomonas strains as a host cell for hGHproduction.

Once a recombinant host cell strain has been established (i.e., theexpression construct has been introduced into the host cell and hostcells with the proper expression construct are isolated), therecombinant host cell strain is cultured under conditions appropriatefor production of hIFN polypeptides. As will be apparent to one of skillin the art, the method of culture of the recombinant host cell strainwill be dependent on the nature of the expression construct utilized andthe identity of the host cell. Recombinant host strains are normallycultured using methods that are known to those of ordinary skill in theart. Recombinant host cells are typically cultured in liquid mediumcontaining assimilatable sources of carbon, nitrogen, and inorganicsalts and, optionally, containing vitamins, amino acids, growth factors,and other proteinaceous culture supplements known to those of ordinaryskill in the art. Liquid media for culture of host cells may optionallycontain antibiotics or anti-fungals to prevent the growth of undesirablemicroorganisms and/or compounds including, but not limited to,antibiotics to select for host cells containing the expression vector.

Recombinant host cells may be cultured in batch or continuous formats,with either cell harvesting (in the case where the hIFN polypeptideaccumulates intracellularly) or harvesting of culture supernatant ineither batch or continuous formats. For production in prokaryotic hostcells, batch culture and cell harvest are preferred.

The hIFN polypeptides of the present invention are normally purifiedafter expression in recombinant systems. Voss et al. Biochem J. (1994)298:719-725; Rosendahl et al. Bioconjugate Chem (2005) 16:200-207;Swaminathan et al. Protein Expression and Purification (1999)15:236-242; and Neves et al. Protein Expression and Purification (2004)35:353-359, which are incorporated by reference herein, describe methodsof expression, purification, isolation and characterization ofrecombinant interferons. The hIFN polypeptide may be purified from hostcells or culture medium by a variety of methods known to the art. hIFNpolypeptides produced in bacterial host cells may be poorly soluble orinsoluble (in the form of inclusion bodies). In one embodiment of thepresent invention, amino acid substitutions may readily be made in thehIFN polypeptide that are selected for the purpose of increasing thesolubility of the recombinantly produced protein utilizing the methodsdisclosed herein as well as those known in the art. In the case ofinsoluble protein, the protein may be collected from host cell lysatesby centrifugation and may further be followed by homogenization of thecells. In the case of poorly soluble protein, compounds including, butnot limited to, polyethylene imine (PEI) may be added to induce theprecipitation of partially soluble protein. The precipitated protein maythen be conveniently collected by centrifugation. Recombinant host cellsmay be disrupted or homogenized to release the inclusion bodies fromwithin the cells using a variety of methods known to those of ordinaryskill in the art. Host cell disruption or homogenization may beperformed using well known techniques including, but not limited to,enzymatic cell disruption, sonication, dounce homogenization, or highpressure release disruption. In one embodiment of the method of thepresent invention, the high pressure release technique is used todisrupt the E. coli host cells to release the inclusion bodies of thehIFN polypeptides. When handling inclusion bodies of hIFN polypeptide,it may be advantageous to minimize the homogenization time onrepetitions in order to maximize the yield of inclusion bodies withoutloss due to factors such as solubilization, mechanical shearing orproteolysis.

Insoluble or precipitated hIFN polypeptide may then be solubilized usingany of a number of suitable solubilization agents known to the art. ThehIFN polypeptide may be solubilized with urea or guanidinehydrochloride. The volume of the solubilized hIFN polypeptide-BP shouldbe minimized so that large batches may be produced using convenientlymanageable batch sizes. This factor may be significant in a large-scalecommercial setting where the recombinant host may be grown in batchesthat are thousands of liters in volume. In addition, when manufacturinghIFN polypeptide in a large-scale commercial setting, in particular forhuman pharmaceutical uses, the avoidance of harsh chemicals that candamage the machinery and container, or the protein product itself,should be avoided, if possible. It has been shown in the method of thepresent invention that the milder denaturing agent urea can be used tosolubilize the hIFN polypeptide inclusion bodies in place of the harsherdenaturing agent guanidine hydrochloride. The use of urea significantlyreduces the risk of damage to stainless steel equipment utilized in themanufacturing and purification process of hIFN polypeptide whileefficiently solubilizing the hIFN polypeptide inclusion bodies.

In the case of soluble hIFN protein, the hIFN may be secreted into theperiplasmic space or into the culture medium. In addition, soluble hIFNmay be present in the cytoplasm of the host cells. It may be desired toconcentrate soluble hIFN prior to performing purification steps.Standard techniques known to those of ordinary skill in the art may beused to concentrate soluble hIFN from, for example, cell lysates orculture medium. In addition, standard techniques known to those ofordinary skill in the art may be used to disrupt host cells and releasesoluble hIFN from the cytoplasm or periplasmic space of the host cells.

When hIFN polypeptide is produced as a fusion protein, the fusionsequence may be removed. Removal of a fusion sequence may beaccomplished by enzymatic or chemical cleavage. Enzymatic removal offusion sequences may be accomplished using methods known to those ofordinary skill in the art. The choice of enzyme for removal of thefusion sequence will be determined by the identity of the fusion, andthe reaction conditions will be specified by the choice of enzyme aswill be apparent to one of ordinary skill in the art. Chemical cleavagemay be accomplished using reagents known to those of ordinary skill inthe art, including but not limited to, cyanogen bromide, TEV protease,and other reagents. The cleaved hIFN polypeptide may be purified fromthe cleaved fusion sequence by methods known to those of ordinary skillin the art. Such methods will be determined by the identity andproperties of the fusion sequence and the hIFN polypeptide, as will beapparent to one of ordinary skill in the art. Methods for purificationmay include, but are not limited to, size-exclusion chromatography,hydrophobic interaction chromatography, ion-exchange chromatography ordialysis or any combination thereof.

The hIFN polypeptide may also be purified to remove DNA from the proteinsolution. DNA may be removed by any suitable method known to the art,such as precipitation or ion exchange chromatography, but may be removedby precipitation with a nucleic acid precipitating agent, such as, butnot limited to, protamine sulfate. The hIFN polypeptide may be separatedfrom the precipitated DNA using standard well known methods including,but not limited to, centrifugation or filtration. Removal of hostnucleic acid molecules is an important factor in a setting where thehIFN polypeptide is to be used to treat humans and the methods of thepresent invention reduce host cell DNA to pharmaceutically acceptablelevels.

Methods for small-scale or large-scale fermentation can also be used inprotein expression, including but not limited to, fermentors, shakeflasks, fluidized bed bioreactors, hollow fiber bioreactors, rollerbottle culture systems, and stirred tank bioreactor systems. Each ofthese methods can be performed in a batch, fed-batch, or continuous modeprocess.

Human hIFN polypeptides of the invention can generally be recoveredusing methods standard in the art. For example, culture medium or celllysate can be centrifuged or filtered to remove cellular debris. Thesupernatant may be concentrated or diluted to a desired volume ordiafiltered into a suitable buffer to condition the preparation forfurther purification. Further purification of the hIFN polypeptide ofthe present invention includes separating deamidated, clipped,acetylated, and oxidized forms of the hIFN polypeptide variant from theintact form.

Any of the following exemplary procedures can be employed forpurification of hIFN polypeptides of the invention: affinitychromatography; anion- or cation-exchange chromatography (using,including but not limited to, DEAE SEPHAROSE); chromatography on silica;High Performance Liquid Chromatography (HPLC); reverse phase HPLC; gelfiltration (using, including but not limited to, SEPHADEX G-75);hydrophobic interaction chromatography; size-exclusion chromatography,metal-chelate chromatography; ultrafiltration/diafiltration; ethanolprecipitation; ammonium sulfate precipitation; chromatofocusing;displacement chromatography; electrophoretic procedures (including butnot limited to preparative isoelectric focusing), differentialsolubility (including but not limited to ammonium sulfateprecipitation), SDS-PAGE, or extraction. “Protein Purification” (1998)Janson et Ryden eds. (A. John Wiley and Sons, Inc. Publishers) describevarious methods of chromatography and electrophoresis as well asmodifications that can be performed within each method for optimizationof purification schemes.

Proteins of the present invention, including but not limited to,proteins comprising unnatural amino acids, antibodies to proteinscomprising unnatural amino acids, binding partners for proteinscomprising unnatural amino acids, etc., can be purified, eitherpartially or substantially to homogeneity, according to standardprocedures known to and used by those of skill in the art. Accordingly,polypeptides of the invention can be recovered and purified by any of anumber of methods known to those of ordinary skill in the art, includingbut not limited to, ammonium sulfate or ethanol precipitation, acid orbase extraction, column chromatography, affinity column chromatography,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, hydroxylapatitechromatography, lectin chromatography, gel electrophoresis and the like.Protein refolding steps can be used, as desired, in making correctlyfolded mature proteins. High performance liquid chromatography (HPLC),affinity chromatography or other suitable methods can be employed infinal purification steps where high purity is desired. In oneembodiment, antibodies made against unnatural amino acids (or proteinscomprising unnatural amino acids) are used as purification reagents,including but not limited to, for affinity-based purification ofproteins comprising one or more unnatural amino acid(s). Once purified,partially or to homogeneity, as desired, the polypeptides are optionallyused for a wide variety of utilities, including but not limited to, asassay components, therapeutics, prophylaxis, diagnostics, researchreagents, and/or as immunogens for antibody production.

In addition to other references noted herein, a variety ofpurification/protein folding methods are known to those of ordinaryskill in the art, including, but not limited to, those set forth in R.Scopes, Protein Purification, Springer-Verlag, N.Y. (1982); Deutscher,Methods in Enzymology Vol. 182: Guide to Protein Purification, AcademicPress, Inc. N.Y. (1990); Sandana, (1997) Bioseparation of Proteins,Academic Press, Inc.; Bollag et al. (1996) Protein Methods, 2nd EditionWiley-Liss, NY; Walker, (1996) The Protein Protocols Handbook HumanaPress, NJ, Harris and Angal, (1990) Protein Purification Applications: APractical Approach IRL Press at Oxford, Oxford, England; Harris andAngal, Protein Purification Methods: A Practical Approach IRL Press atOxford, Oxford, England; Scopes, (1993) Protein Purification: Principlesand Practice 3rd Edition Springer Verlag, NY; Janson and Ryden, (1998)Protein Purification: Principles, High Resolution Methods andApplications, Second Edition Wiley-VCH, NY; and Walker (1998), ProteinProtocols on CD-ROM Humana Press, NJ; and the references cited therein.

One advantage of producing a protein or polypeptide of interest with anunnatural amino acid in a eukaryotic host cell or non-eukaryotic hostcell is that typically the proteins or polypeptides will be folded intheir native conformations. However, in certain embodiments of theinvention, those of skill in the art will recognize that, aftersynthesis, expression and/or purification, proteins can possess aconformation different from the desired conformations of the relevantpolypeptides. In one aspect of the invention, the expressed protein orpolypeptide is optionally denatured and then renatured. This isaccomplished utilizing methods known in the art, including but notlimited to, by adding a chaperonin to the protein or polypeptide ofinterest, by solubilizing the proteins in a chaotropic agent such asguanidine HCl, utilizing protein disulfide isomerase, etc.

In general, it is occasionally desirable to denature and reduceexpressed polypeptides and then to cause the polypeptides to re-foldinto the preferred conformation. For example, guanidine, urea, DTT, DTE,and/or a chaperonin can be added to a translation product of interest.Methods of reducing, denaturing and renaturing proteins are known tothose of ordinary skill in the art (see, the references above, andDebinski, et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman andPastan (1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al., (1992)Anal. Biochem., 205: 263-270). Debinski, et al., for example, describethe denaturation and reduction of inclusion body proteins inguanidine-DTE. The proteins can be refolded in a redox buffercontaining, including but not limited to, oxidized glutathione andL-arginine. Refolding reagents can be flowed or otherwise moved intocontact with the one or more polypeptide or other expression product, orvice-versa.

In the case of prokaryotic production of hIFN polypeptide, the hIFNpolypeptide thus produced may be misfolded and thus lacks or has reducedbiological activity. The bioactivity of the protein may be restored by“refolding”. In general, misfolded hIFN polypeptide is refolded bysolubilizing (where the hIFN polypeptide is also insoluble), unfoldingand reducing the polypeptide chain using, for example, one or morechaotropic agents (e.g. urea and/or guanidine) and a reducing agentcapable of reducing disulfide bonds (e.g. dithiothreitol, DTT or2-mercaptoethanol, 2-ME). At a moderate concentration of chaotrope, anoxidizing agent is then added (e.g., oxygen, cystine or cystamine),which allows the reformation of disulfide bonds. hIFN polypeptide may berefolded using standard methods known in the art, such as thosedescribed in U.S. Pat. Nos. 4,511,502, 4,511,503, and 4,512,922, whichare incorporated by reference herein. The hIFN polypeptide may also becofolded with other proteins to form heterodimers or heteromultimers.

After refolding, the hIFN may be further purified. Purification of hIFNmay be accomplished using a variety of techniques known to those ofordinary skill in the art, including hydrophobic interactionchromatography, size exclusion chromatography, ion exchangechromatography, reverse-phase high performance liquid chromatography,affinity chromatography, and the like or any combination thereof.Additional purification may also include a step of drying orprecipitation of the purified protein.

After purification, hIFN may be exchanged into different buffers and/orconcentrated by any of a variety of methods known to the art, including,but not limited to, diafiltration and dialysis. hIFN that is provided asa single purified protein may be subject to aggregation andprecipitation.

The purified hIFN may be at least 90% pure (as measured by reverse phasehigh performance liquid chromatography, RP-HPLC, or sodium dodecylsulfate-polyacrylamide gel electrophoresis, SDS-PAGE) or at least 95%pure, or at least 98% pure, or at least 99% or greater pure. Regardlessof the exact numerical value of the purity of the hIFN, the hIFN issufficiently pure for use as a pharmaceutical product or for furtherprocessing, such as conjugation with a water soluble polymer such asPEG.

Certain hIFN molecules may be used as therapeutic agents in the absenceof other active ingredients or proteins (other than excipients,carriers, and stabilizers, serum albumin and the like), or they may becomplexed with another protein or a polymer.

General Purification Methods Any one of a variety of isolation steps maybe performed on the cell lysate, extract, culture medium, inclusionbodies, periplasmic space of the host cells, cytoplasm of the hostcells, or other material, comprising hIFN polypeptide or on any hIFNpolypeptide mixtures resulting from any isolation steps including, butnot limited to, affinity chromatography, ion exchange chromatography,hydrophobic interaction chromatography, gel filtration chromatography,high performance liquid chromatography (“HPLC”), reversed phase-HPLC(“RP-HPLC”), expanded bed adsorption, or any combination and/orrepetition thereof and in any appropriate order.

Equipment and other necessary materials used in performing thetechniques described herein are commercially available. Pumps, fractioncollectors, monitors, recorders, and entire systems are available from,for example, Applied Biosystems (Foster City, Calif.), Bio-RadLaboratories, Inc. (Hercules, Calif.), and Amersham Biosciences, Inc.(Piscataway, N.J.). Chromatographic materials including, but not limitedto, exchange matrix materials, media, and buffers are also availablefrom such companies.

Equilibration, and other steps in the column chromatography processesdescribed herein such as washing and elution, may be more rapidlyaccomplished using specialized equipment such as a pump. Commerciallyavailable pumps include, but are not limited to, HILOAD® Pump P-50,Peristaltic Pump P-1, Pump P-901, and Pump P-903 (Amersham Biosciences,Piscataway, N.J.).

Examples of fraction collectors include RediFrac Fraction Collector,FRAC-100 and FRAC-200 Fraction Collectors, and SUPERFRAC® FractionCollector (Amersham Biosciences, Piscataway, N.J.). Mixers are alsoavailable to form pH and linear concentration gradients. Commerciallyavailable mixers include Gradient Mixer GM-1 and In-Line Mixers(Amersham Biosciences, Piscataway, N.J.).

The chromatographic process may be monitored using any commerciallyavailable monitor. Such monitors may be used to gather information likeUV, pH, and conductivity. Examples of detectors include Monitor UV-1,UVICORD® S II, Monitor UV-M II, Monitor UV-900, Monitor UPC-900, MonitorpH/C-900, and Conductivity Monitor (Amersham Biosciences, Piscataway,N.J.). Indeed, entire systems are commercially available including thevarious AKTA® systems from Amersham Biosciences (Piscataway, N.J.).

In one embodiment of the present invention, for example, the hIFNpolypeptide may be reduced and denatured by first denaturing theresultant purified hIFN polypeptide in urea, followed by dilution intoTRIS buffer containing a reducing agent (such as DTT) at a suitable pH.In another embodiment, the hIFN polypeptide is denatured in urea in aconcentration range of between about 2 M to about 9 M, followed bydilution in TRIS buffer at a pH in the range of about 5.0 to about 8.0.The refolding mixture of this embodiment may then be incubated. In oneembodiment, the refolding mixture is incubated at room temperature forfour to twenty-four hours. The reduced and denatured hIFN polypeptidemixture may then be further isolated or purified.

As stated herein, the pH of the first hIFN polypeptide mixture may beadjusted prior to performing any subsequent isolation steps. Inaddition, the first hIFN polypeptide mixture or any subsequent mixturethereof may be concentrated using techniques known in the art. Moreover,the elution buffer comprising the first hIFN polypeptide mixture or anysubsequent mixture thereof may be exchanged for a buffer suitable forthe next isolation step using techniques known to those of ordinaryskill in the art.

Ion Exchange Chromatography In one embodiment, and as an optional,additional step, ion exchange chromatography may be performed on thefirst hIFN polypeptide mixture. See generally ION EXCHANGECHROMATOGRAPHY: PRINCIPLES AND METHODS (Cat. No. 18-1114-21, AmershamBiosciences (Piscataway, N.J.)). Commercially available ion exchangecolumns include HITRAP®, HIPREP®, and HILOAD® Columns (AmershamBiosciences, Piscataway, N.J.). Such columns utilize strong anionexchangers such as Q SEPHAROSE® Fast Flow, Q SEPHAROSE® HighPerformance, and Q SEPHAROSE® XL; strong cation exchangers such as SPSEPHAROSE® High Performance, SP SEPHAROSE® Fast Flow, and SP SEPHAROSE®XL; weak anion exchangers such as DEAE SEPHAROSE® Fast Flow; and weakcation exchangers such as CM SEPHAROSE® Fast Flow (Amersham Biosciences,Piscataway, N.J.). Anion or cation exchange column chromatography may beperformed on the hIFN polypeptide at any stage of the purificationprocess to isolate substantially purified hIFN polypeptide. The cationexchange chromatography step may be performed using any suitable cationexchange matrix. Useful cation exchange matrices include, but are notlimited to, fibrous, porous, non-porous, microgranular, beaded, orcross-linked cation exchange matrix materials. Such cation exchangematrix materials include, but are not limited to, cellulose, agarose,dextran, polyacrylate, polyvinyl, polystyrene, silica, polyether, orcomposites of any of the foregoing.

Following adsorption of the hIFN polypeptide to the cation exchangermatrix, substantially purified hIFN polypeptide may be eluted bycontacting the matrix with a buffer having a sufficiently high pH orionic strength to displace the hIFN polypeptide from the matrix.Suitable buffers for use in high pH elution of substantially purifiedhIFN polypeptide include, but are not limited to, citrate, phosphate,formate, acetate, HEPES, and MES buffers ranging in concentration fromat least about 5 mM to at least about 100 mM.

Reverse-Phase Chromatography RP-HPLC may be performed to purify proteinsfollowing suitable protocols that are known to those of ordinary skillin the art. See, e.g., Pearson et al., ANAL BIOCHEM. (1982) 124:217-230(1982); Rivier et al., J. CHROM. (1983) 268:112-119; Kunitani et al., J.CHROM. (1986) 359:391-402. RP-HPLC may be performed on the hIFNpolypeptide to isolate substantially purified hIFN polypeptide. In thisregard, silica derivatized resins with alkyl functionalities with a widevariety of lengths, including, but not limited to, at least about C₃ toat least about C₃₀, at least about C₃ to at least about C₂₀, or at leastabout C₃ to at least about C₁₈, resins may be used. Alternatively, apolymeric resin may be used. For example, TosoHaas Amberchrome CG1000sdresin may be used, which is a styrene polymer resin. Cyano or polymericresins with a wide variety of alkyl chain lengths may also be used.Furthermore, the RP-HPLC column may be washed with a solvent such asethanol. The Source RP column is another example of a RP-HPLC column.

A suitable elution buffer containing an ion pairing agent and an organicmodifier such as methanol, isopropanol, tetrahydrofuran, acetonitrile orethanol, may be used to elute the hIFN polypeptide from the RP-HPLCcolumn. The most commonly used ion pairing agents include, but are notlimited to, acetic acid, formic acid, perchloric acid, phosphoric acid,trifluoroacetic acid, heptafluorobutyric acid, triethylamine,tetramethylammonium, tetrabutylammonium, triethylammonium acetate.Elution may be performed using one or more gradients or isocraticconditions, with gradient conditions preferred to reduce the separationtime and to decrease peak width. Another method involves the use of twogradients with different solvent concentration ranges. Examples ofsuitable elution buffers for use herein may include, but are not limitedto, ammonium acetate and acetonitrile solutions.

Hydrophobic Interaction Chromatography Purification TechniquesHydrophobic interaction chromatography (HIC) may be performed on thehIFN polypeptide. See generally HYDROPHOBIC INTERACTION CHROMATOGRAPHYHANDBOOK: PRINCIPLES AND METHODS (Cat. No. 18-1020-90, AmershamBiosciences (Piscataway, N.J.) which is incorporated by referenceherein. Suitable HIC matrices may include, but are not limited to,alkyl- or aryl-substituted matrices, such as butyl-, hexyl-, octyl- orphenyl-substituted matrices including agarose, cross-linked agarose,sepharose, cellulose, silica, dextran, polystyrene, poly(methacrylate)matrices, and mixed mode resins, including but not limited to, apolyethyleneamine resin or a butyl- or phenyl-substitutedpoly(methacrylate) matrix. Commercially available sources forhydrophobic interaction column chromatography include, but are notlimited to, HITRAP®, HIPREP®, and HILOAD® columns (Amersham Biosciences,Piscataway, N.J.).

Briefly, prior to loading, the HIC column may be equilibrated usingstandard buffers known to those of ordinary skill in the art, such as anacetic acid/sodium chloride solution or HEPES containing ammoniumsulfate. Ammonium sulfate may be used as the buffer for loading the HICcolumn. After loading the hIFN polypeptide, the column may then washedusing standard buffers and conditions to remove unwanted materials butretaining the hIFN polypeptide on the HIC column. The hIFN polypeptidemay be eluted with about 3 to about 10 column volumes of a standardbuffer, such as a HEPES buffer containing EDTA and lower ammoniumsulfate concentration than the equilibrating buffer, or an aceticacid/sodium chloride buffer, among others. A decreasing linear saltgradient using, for example, a gradient of potassium phosphate, may alsobe used to elute the hIFN molecules. The eluant may then beconcentrated, for example, by filtration such as diafiltration orultrafiltration. Diafiltration may be utilized to remove the salt usedto elute the hIFN polypeptide.

Other Purification Techniques Yet another isolation step using, forexample, gel filtration (GEL FILTRATION: PRINCIPLES AND METHODS (Cat.No. 18-1022-18, Amersham Biosciences, Piscataway, N.J.) which isincorporated by reference herein, hydroxyapatite chromatography(suitable matrices include, but are not limited to, HA-Ultrogel, HighResolution (Calbiochem), CHT Ceramic Hydroxyapatite (BioRad), Bio—GelHTP Hydroxyapatite (BioRad)), HPLC, expanded bed adsorption,ultrafiltration, diafiltration, lyophilization, and the like, may beperformed on the first hIFN polypeptide mixture or any subsequentmixture thereof, to remove any excess salts and to replace the bufferwith a suitable buffer for the next isolation step or even formulationof the final drug product.

The yield of hIFN polypeptide, including substantially purified hIFNpolypeptide, may be monitored at each step described herein usingtechniques known to those of ordinary skill in the art. Such techniquesmay also be used to assess the yield of substantially purified hIFNpolypeptide following the last isolation step. For example, the yield ofhIFN polypeptide may be monitored using any of several reverse phasehigh pressure liquid chromatography columns, having a variety of alkylchain lengths such as cyano RP-HPLC, Cl₈RP-HPLC; as well as cationexchange HPLC and gel filtration HPLC.

In specific embodiments of the present invention, the yield of hIFNafter each purification step may be at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, at least about99.9%, or at least about 99.99%, of the hIFN in the starting materialfor each purification step.

Purity may be determined using standard techniques, such as SDS-PAGE, orby measuring hIFN polypeptide using Western blot and ELISA assays. Forexample, polyclonal antibodies may be generated against proteinsisolated from negative control yeast fermentation and the cationexchange recovery. The antibodies may also be used to probe for thepresence of contaminating host cell proteins.

RP-HPLC material Vydac C4 (Vydac) consists of silica gel particles, thesurfaces of which carry C4-alkyl chains. The separation of hIFNpolypeptide from the proteinaceous impurities is based on differences inthe strength of hydrophobic interactions. Elution is performed with anacetonitrile gradient in diluted trifluoroacetic acid. Preparative HPLCis performed using a stainless steel column (filled with 2.8 to 3.2liter of Vydac C4 silicagel). The Hydroxyapatite Ultrogel eluate isacidified by adding trifluoroacetic acid and loaded onto the Vydac C4column. For washing and elution an acetonitrile gradient in dilutedtrifluoroacetic acid is used. Fractions are collected and immediatelyneutralized with phosphate buffer. The hIFN polypeptide fractions whichare within the IPC limits are pooled.

DEAE Sepharose (Pharmacia) material consists of diethylaminoethyl(DEAE)-groups which are covalently bound to the surface of Sepharosebeads. The binding of hIFN polypeptide to the DEAE groups is mediated byionic interactions. Acetonitrile and trifluoroacetic acid pass throughthe column without being retained. After these substances have beenwashed off, trace impurities are removed by washing the column withacetate buffer at a low pH. Then the column is washed with neutralphosphate buffer and hIFN polypeptide is eluted with a buffer withincreased ionic strength. The column is packed with DEAE Sepharose fastflow. The column volume is adjusted to assure a hIFN polypeptide load inthe range of 3-10 mg hIFN polypeptide/ml gel. The column is washed withwater and equilibration buffer (sodium/potassium phosphate). The pooledfractions of the HPLC eluate are loaded and the column is washed withequilibration buffer. Then the column is washed with washing buffer(sodium acetate buffer) followed by washing with equilibration buffer.Subsequently, hIFN polypeptide is eluted from the column with elutionbuffer (sodium chloride, sodium/potassium phosphate) and collected in asingle fraction in accordance with the master elution profile. Theeluate of the DEAE Sepharose column is adjusted to the specifiedconductivity. The resulting drug substance is sterile filtered intoTeflon bottles and stored at −70° C.

Additional methods that may be employed include, but are not limited to,steps to remove endotoxins. Endotoxins are lipopoly-saccharides (LPSs)which are located on the outer membrane of Gram-negative host cells,such as, for example, Escherichia coli. Methods for reducing endotoxinlevels are known to one of ordinary skill in the art and include, butare not limited to, purification techniques using silica supports, glasspowder or hydroxyapatite, reverse-phase, affinity, size-exclusion,anion-exchange chromatography, hydrophobic interaction chromatography, acombination of these methods, and the like. Modifications or additionalmethods may be required to remove contaminants such as co-migratingproteins from the polypeptide of interest. Methods for measuringendotoxin levels are known to one of ordinary skill in the art andinclude, but are not limited to, Limulus Amebocyte Lysate (LAL) assays.The Endosafe™-PTS assay is a colorimetric, single tube system thatutilizes cartridges preloaded with LAL reagent, chromogenic substrate,and control standard endotoxin along with a handheld spectrophotometer.Alternate methods include, but are not limited to, a Kinetic LAL methodthat is turbidimetric and uses a 96 well format.

A wide variety of methods and procedures can be used to assess the yieldand purity of a hIFN protein comprising one or more non-naturallyencoded amino acids, including but not limited to, the Bradford assay,SDS-PAGE, silver stained SDS-PAGE, coomassie stained SDS-PAGE, massspectrometry (including but not limited to, MALDI-TOF) and other methodsfor characterizing proteins known to one of ordinary skill in the art.

Additional methods include, but are not limited to: SDS-PAGE coupledwith protein staining methods, immunoblotting, matrix assisted laserdesorption/ionization-mass spectrometry (MALDI-MS), liquidchromatography/mass spectrometry, isoelectric focusing, analytical anionexchange, chromatofocusing, and circular dichroism.

VIII. Expression in Alternate Systems

Several strategies have been employed to introduce unnatural amino acidsinto proteins in non-recombinant host cells, mutagenized host cells, orin cell-free systems. These systems are also suitable for use in makingthe hIFN polypeptides of the present invention. Derivatization of aminoacids with reactive side-chains such as Lys, Cys and Tyr resulted in theconversion of lysine to N²-acetyl-lysine. Chemical synthesis alsoprovides a straightforward method to incorporate unnatural amino acids.With the recent development of enzymatic ligation and native chemicalligation of peptide fragments, it is possible to make larger proteins.See, e.g., P. E. Dawson and S. B. H. Kent, Annu. Rev. Biochem, 69:923(2000). Chemical peptide ligation and native chemical ligation aredescribed in U.S. Pat. No. 6,184,344, U.S. Patent Publication No.2004/0138412, U.S. Patent Publication No. 2003/0208046, WO 02/098902,and WO 03/042235, which are incorporated by reference herein. A generalin vitro biosynthetic method in which a suppressor tRNA chemicallyacylated with the desired unnatural amino acid is added to an in vitroextract capable of supporting protein biosynthesis, has been used tosite-specifically incorporate over 100 unnatural amino acids into avariety of proteins of virtually any size. See, e.g., V. W. Cornish, D.Mendel and P. G. Schultz, Angew. Chem. Int. Ed. Engl., 1995, 34:621(1995); C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, P. G.Schultz, A general method for site-specific incorporation of unnaturalamino acids into proteins, Science 244:182-188 (1989); and, J. D. Bain,C. G. Glabe, T. A. Dix, A. R. Chamberlin, E. S. Diala, Biosyntheticsite-specific incorporation of a non-natural amino acid into apolypeptide, J. Am. Chem. Soc. 111:8013-8014 (1989). A broad range offunctional groups has been introduced into proteins for studies ofprotein stability, protein folding, enzyme mechanism, and signaltransduction.

An in vivo method, termed selective pressure incorporation, wasdeveloped to exploit the promiscuity of wild-type synthetases. See,e.g., N. Budisa, C. Minks, S. Alefelder, W. Wenger, F. M. Dong, L.Moroder and R. Huber, FASEB J., 13:41 (1999). An auxotrophic strain, inwhich the relevant metabolic pathway supplying the cell with aparticular natural amino acid is switched off, is grown in minimal mediacontaining limited concentrations of the natural amino acid, whiletranscription of the target gene is repressed. At the onset of astationary growth phase, the natural amino acid is depleted and replacedwith the unnatural amino acid analog. Induction of expression of therecombinant protein results in the accumulation of a protein containingthe unnatural analog. For example, using this strategy, o, m andp-fluorophenylalanines have been incorporated into proteins, and exhibittwo characteristic shoulders in the UV spectrum which can be easilyidentified, see, e.g., C. Minks, R. Huber, L. Moroder and N. Budisa,Anal. Biochem., 284:29 (2000); trifluoromethionine has been used toreplace methionine in bacteriophage T4 lysozyme to study its interactionwith chitooligosaccharide ligands by ¹⁹F NMR, see, e.g., H. Duewel, E.Daub, V. Robinson and J. F. Honek, Biochemistry, 36:3404 (1997); andtrifluoroleucine has been incorporated in place of leucine, resulting inincreased thermal and chemical stability of a leucine-zipper protein.See, e.g., Y. Tang, G. Ghirlanda, W. A. Petka, T. Nakajima, W. F.DeGrado and D. A. Tirrell, Angew. Chem. Int. Ed. Engl., 40:1494 (2001).Moreover, selenomethionine and telluromethionine are incorporated intovarious recombinant proteins to facilitate the solution of phases inX-ray crystallography. See, e.g., W. A. Hendrickson, J. R. Horton and D.M. Lemaster, EMBO J., 9:1665 (1990); J. O. Boles, K. Lewinski, M.Kunkle, J. D. Odom, B. Dunlap, L. Lebioda and M. Hatada, Nat. Struct.Biol., 1:283 (1994); N. Budisa, B. Steipe, P. Demange, C. Eckerskorn, J.Kellermann and R. Huber, Eur. J. Biochem., 230:788 (1995); and, N.Budisa, W. Karnbrock, S. Steinbacher, A. Humm, L. Prade, T. Neuefeind,L. Moroder and R. Huber, J. Mol. Biol., 270:616 (1997). Methionineanalogs with alkene or alkyne functionalities have also beenincorporated efficiently, allowing for additional modification ofproteins by chemical means. See, e.g., J. C. van Hest and D. A. Tirrell,FEBS Lett., 428:68 (1998); J. C. van Hest, K. L. Kiick and D. A.Tirrell, J. Am. Chem. Soc., 122:1282 (2000); and, K. L. Kiick and D. A.Tirrell, Tetrahedron, 56:9487 (2000); U.S. Pat. No. 6,586,207; U.S.Patent Publication 2002/0042097, which are incorporated by referenceherein.

The success of this method depends on the recognition of the unnaturalamino acid analogs by aminoacyl-tRNA synthetases, which, in general,require high selectivity to insure the fidelity of protein translation.One way to expand the scope of this method is to relax the substratespecificity of aminoacyl-tRNA synthetases, which has been achieved in alimited number of cases. For example, replacement of Ala²⁹⁴ by Gly inEscherichia coli phenylalanyl-tRNA synthetase (PheRS) increases the sizeof substrate binding pocket, and results in the acylation of tRNAPhe byp-Cl-phenylalanine (p-Cl-Phe). See, M. Ibba, P. Kast and H. Hennecke,Biochemistry, 33:7107 (1994). An Escherichia coli strain harboring thismutant PheRS allows the incorporation of p-Cl-phenylalanine orp-Br-phenylalanine in place of phenylalanine See, e.g., M. Ibba and H.Hennecke, FEBS Lett., 364:272 (1995); and, N. Sharma, R. Furter, P. Kastand D. A. Tirrell, FEBS Lett., 467:37 (2000). Similarly, a pointmutation Phe130Ser near the amino acid binding site of Escherichia colityrosyl-tRNA synthetase was shown to allow azatyrosine to beincorporated more efficiently than tyrosine. See, F. Hamano-Takaku, T.Iwama, S. Saito-Yano, K. Takaku, Y. Monden, M. Kitabatake, D. Soll andS. Nishimura, J. Biol. Chem., 275:40324 (2000).

Another strategy to incorporate unnatural amino acids into proteins invivo is to modify synthetases that have proofreading mechanisms. Thesesynthetases cannot discriminate and therefore activate amino acids thatare structurally similar to the cognate natural amino acids. This erroris corrected at a separate site, which deacylates the mischarged aminoacid from the tRNA to maintain the fidelity of protein translation. Ifthe proofreading activity of the synthetase is disabled, structuralanalogs that are misactivated may escape the editing function and beincorporated. This approach has been demonstrated recently with thevalyl-tRNA synthetase (ValRS). See, V. Doring, H. D. Mootz, L. A.Nangle, T. L. Hendrickson, V. de Crecy-Lagard, P. Schimmel and P.Marliere, Science, 292:501 (2001). ValRS can misaminoacylate tRNAValwith Cys, Thr, or aminobutyrate (Abu); these noncognate amino acids aresubsequently hydrolyzed by the editing domain. After random mutagenesisof the Escherichia coli chromosome, a mutant Escherichia coli strain wasselected that has a mutation in the editing site of ValRS. Thisedit-defective ValRS incorrectly charges tRNAVal with Cys. Because Abusterically resembles Cys (—SH group of Cys is replaced with —CH3 inAbu), the mutant ValRS also incorporates Abu into proteins when thismutant Escherichia coli strain is grown in the presence of Abu. Massspectrometric analysis shows that about 24% of valines are replaced byAbu at each valine position in the native protein.

Solid-phase synthesis and semisynthetic methods have also allowed forthe synthesis of a number of proteins containing novel amino acids. Forexample, see the following publications and references cited within,which are as follows: Crick, F. H. C., Barrett, L. Brenner, S.Watts-Tobin, R. General nature of the genetic code for proteins. Nature,192:1227-1232 (1961); Hofmann, K., Bohn, H. Studies on polypeptides.XXVI. The effect of pyrazole-imidazole replacements on the S-proteinactivating potency of an S-peptide fragment, J. Am. Chem,88(24):5914-5919 (1966); Kaiser, E. T. Synthetic approaches tobiologically active peptides and proteins including enzymes, Acc ChemRes, 22:47-54 (1989); Nakatsuka, T., Sasaki, T., Kaiser, E. T. Peptidesegment coupling catalyzed by the semisynthetic enzyme thiosubtilisin, JAm Chem Soc, 109:3808-3810 (1987); Schnolzer, M., Kent, S B H.Constructing proteins by dovetailing unprotected synthetic peptides:backbone-engineered HIV protease, Science, 256(5054):221-225 (1992);Chaiken, I. M. Semisynthetic peptides and proteins, CRC Crit RevBiochem, 11(3):255-301 (1981); Offord, R. E. Protein engineering bychemical means? Protein Eng., 1(3):151-157 (1987); and, Jackson, D. Y.,Burnier, J., Quan, C., Stanley, M., Tom, J., Wells, J. A. A DesignedPeptide Ligase for Total Synthesis of Ribonuclease A with UnnaturalCatalytic Residues, Science, 266(5183):243 (1994).

Chemical modification has been used to introduce a variety of unnaturalside chains, including cofactors, spin labels and oligonucleotides intoproteins in vitro. See, e.g., Corey, D. R., Schultz, P. G. Generation ofa hybrid sequence-specific single-stranded deoxyribonuclease, Science,238(4832):1401-1403 (1987); Kaiser, E. T., Lawrence D. S., Rokita, S. E.The chemical modification of enzymatic specificity, Annu Rev Biochem,54:565-595 (1985); Kaiser, E. T., Lawrence, D. S. Chemical mutation ofenzyme active sites, Science, 226(4674):505-511 (1984); Neet, K. E.,Nanci A, Koshland, D. E. Properties of thiol-subtilisin, J Biol. Chem.,243(24):6392-6401 (1968); Polgar, L. B. et M. L. Bender, A new enzymecontaining a synthetically formed active site. Thiol-subtilisin. J. Am.Chem Soc, 88:3153-3154 (1966); and, Pollack, S. J., Nakayama, G.Schultz, P. G. Introduction of nucleophiles and spectroscopic probesinto antibody combining sites, Science, 242(4881):1038-1040 (1988).

Alternatively, biosynthetic methods that employ chemically modifiedaminoacyl-tRNAs have been used to incorporate several biophysical probesinto proteins synthesized in vitro. See the following publications andreferences cited within: Brunner, J. New Photolabeling and crosslinkingmethods, Annu. Rev Biochem, 62:483-514 (1993); and, Krieg, U. C.,Walter, P., Hohnson, A. E. Photocrosslinking of the signal sequence ofnascent preprolactin of the 54-kilodalton polypeptide of the signalrecognition particle, Proc. Natl. Acad. Sci, 83(22):8604-8608 (1986).

Previously, it has been shown that unnatural amino acids can besite-specifically incorporated into proteins in vitro by the addition ofchemically aminoacylated suppressor tRNAs to protein synthesis reactionsprogrammed with a gene containing a desired amber nonsense mutation.Using these approaches, one can substitute a number of the common twentyamino acids with close structural homologues, e.g., fluorophenylalaninefor phenylalanine, using strains auxotropic for a particular amino acid.See, e.g., Noren, C. J., Anthony-Cahill, Griffith, M. C., Schultz, P. G.A general methodfor site-specific incorporation of unnatural amino acidsinto proteins, Science, 244: 182-188 (1989); M. W. Nowak, et al.,Science 268:439-42 (1995); Bain, J. D., Glabe, C. G., Dix, T. A.,Chamberlin, A. R., Diala, E. S. Biosynthetic site-specific Incorporationof a non-natural amino acid into a polypeptide, J. Am. Chem Soc,111:8013-8014 (1989); N. Budisa et al., FASEB J. 13:41-51 (1999);Ellman, J. A., Mendel, D., Anthony-Cahill, S., Noren, C. J., Schultz, P.G. Biosynthetic methodfor introducing unnatural amino acidssite-specifically into proteins, Methods in Enz., vol. 202, 301-336(1992); and, Mendel, D., Cornish, V. W. & Schultz, P. G. Site-DirectedMutagenesis with an Expanded Genetic Code, Annu Rev Biophys. BiomolStruct. 24, 435-62 (1995).

For example, a suppressor tRNA was prepared that recognized the stopcodon UAG and was chemically aminoacylated with an unnatural amino acid.Conventional site-directed mutagenesis was used to introduce the stopcodon TAG, at the site of interest in the protein gene. See, e.g.,Sayers, J. R., Schmidt, W. Eckstein, F. 5′-3′ Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis, NucleicAcids Res, 16(3):791-802 (1988). When the acylated suppressor tRNA andthe mutant gene were combined in an in vitro transcription/translationsystem, the unnatural amino acid was incorporated in response to the UAGcodon which gave a protein containing that amino acid at the specifiedposition. Experiments using [³H]-Phe and experiments with α-hydroxyacids demonstrated that only the desired amino acid is incorporated atthe position specified by the UAG codon and that this amino acid is notincorporated at any other site in the protein. See, e.g., Noren, et al,supra; Kobayashi et al., (2003) Nature Structural Biology 10(6):425-432;and, Ellman, J. A., Mendel, D., Schultz, P. G. Site-specificincorporation of novel backbone structures into proteins, Science,255(5041):197-200 (1992).

A tRNA may be aminoacylated with a desired amino acid by any method ortechnique, including but not limited to, chemical or enzymaticaminoacylation.

Aminoacylation may be accomplished by aminoacyl tRNA synthetases or byother enzymatic molecules, including but not limited to, ribozymes. Theterm “ribozyme” is interchangeable with “catalytic RNA.” Cech andcoworkers (Cech, 1987, Science, 236:1532-1539; McCorkle et al., 1987,Concepts Biochem. 64:221-226) demonstrated the presence of naturallyoccurring RNAs that can act as catalysts (ribozymes). However, althoughthese natural RNA catalysts have only been shown to act on ribonucleicacid substrates for cleavage and splicing, the recent development ofartificial evolution of ribozymes has expanded the repertoire ofcatalysis to various chemical reactions. Studies have identified RNAmolecules that can catalyze aminoacyl-RNA bonds on their own(2′)3′-termini (Illangakekare et al., 1995 Science 267:643-647), and anRNA molecule which can transfer an amino acid from one RNA molecule toanother (Lohse et al., 1996, Nature 381:442-444).

U.S. Patent Application Publication 2003/0228593, which is incorporatedby reference herein, describes methods to construct ribozymes and theiruse in aminoacylation of tRNAs with naturally encoded and non-naturallyencoded amino acids. Substrate-immobilized forms of enzymatic moleculesthat can aminoacylate tRNAs, including but not limited to, ribozymes,may enable efficient affinity purification of the aminoacylatedproducts. Examples of suitable substrates include agarose, sepharose,and magnetic beads. The production and use of a substrate-immobilizedform of ribozyme for aminoacylation is described in Chemistry andBiology 2003, 10:1077-1084 and U.S. Patent Application Publication2003/0228593, which are incorporated by reference herein.

Chemical aminoacylation methods include, but are not limited to, thoseintroduced by Hecht and coworkers (Hecht, S. M. Ace. Chem. Res. 1992,25, 545; Heckler, T. G.; Roesser, J. R.; Xu, C.; Chang, P.; Hecht, S. M.Biochemistry 1988, 27, 7254; Hecht, S. M.; Alford, B. L.; Kuroda, Y.;Kitano, S. J. Biol. Chem. 1978, 253, 4517) and by Schultz, Chamberlin,Dougherty and others (Cornish, V. W.; Mendel, D.; Schultz, P. G. Angew.Chem. Int. Ed. Engl. 1995, 34, 621; Robertson, S. A.; Ellman, J. A.;Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 2722; Noren, C. J.;Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. Science 1989,244, 182; Bain, J. D.; Glabe, C. G.; Dix, T. A.; Chamberlin, A. R. J.Am. Chem. Soc. 1989, 111, 8013; Bain, J. D. et al. Nature 1992, 356,537; Gallivan, J. P.; Lester, H. A.; Dougherty, D. A. Chem. Biol. 1997,4, 740; Turcatti, et al. J. Biol. Chem. 1996, 271, 19991; Nowak, M. W.et al. Science, 1995, 268, 439; Saks, M. E. et al. J. Biol. Chem. 1996,271, 23169; Hohsaka, T. et al. J. Am. Chem. Soc. 1999, 121, 34), whichare incorporated by reference herein, to avoid the use of synthetases inaminoacylation. Such methods or other chemical aminoacylation methodsmay be used to aminoacylate tRNA molecules.

Methods for generating catalytic RNA may involve generating separatepools of randomized ribozyme sequences, performing directed evolution onthe pools, screening the pools for desirable aminoacylation activity,and selecting sequences of those ribozymes exhibiting desiredaminoacylation activity.

Ribozymes can comprise motifs and/or regions that facilitate acylationactivity, such as a GGU motif and a U-rich region. For example, it hasbeen reported that U-rich regions can facilitate recognition of an aminoacid substrate, and a GGU-motif can form base pairs with the 3′ terminiof a tRNA. In combination, the GGU and motif and U-rich regionfacilitate simultaneous recognition of both the amino acid and tRNAsimultaneously, and thereby facilitate aminoacylation of the 3′ terminusof the tRNA.

Ribozymes can be generated by in vitro selection using a partiallyrandomized r24mini conjugated with tRNA^(Asn) _(CCCG), followed bysystematic engineering of a consensus sequence found in the activeclones. An exemplary ribozyme obtained by this method is termed “Fx3ribozyme” and is described in U.S. Pub. App. No. 2003/0228593, thecontents of which is incorporated by reference herein, acts as aversatile catalyst for the synthesis of various aminoacyl-tRNAs chargedwith cognate non-natural amino acids.

Immobilization on a substrate may be used to enable efficient affinitypurification of the aminoacylated tRNAs. Examples of suitable substratesinclude, but are not limited to, agarose, sepharose, and magnetic beads.Ribozymes can be immobilized on resins by taking advantage of thechemical structure of RNA, such as the 3′-cis-diol on the ribose of RNAcan be oxidized with periodate to yield the corresponding dialdehyde tofacilitate immobilization of the RNA on the resin. Various types ofresins can be used including inexpensive hydrazide resins whereinreductive amination makes the interaction between the resin and theribozyme an irreversible linkage. Synthesis of aminoacyl-tRNAs can besignificantly facilitated by this on-column aminoacylation technique.Kourouklis et al. Methods 2005; 36:239-4 describe a column-basedaminoacylation system.

Isolation of the aminoacylated tRNAs can be accomplished in a variety ofways. One suitable method is to elute the aminoacylated tRNAs from acolumn with a buffer such as a sodium acetate solution with 10 mM EDTA,a buffer containing 50 mMN-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid), 12.5 mM KCl,pH 7.0, 10 mM EDTA, or simply an EDTA buffered water (pH 7.0).

The aminoacylated tRNAs can be added to translation reactions in orderto incorporate the amino acid with which the tRNA was aminoacylated in aposition of choice in a polypeptide made by the translation reaction.Examples of translation systems in which the aminoacylated tRNAs of thepresent invention may be used include, but are not limited to celllysates. Cell lysates provide reaction components necessary for in vitrotranslation of a polypeptide from an input mRNA. Examples of suchreaction components include but are not limited to ribosomal proteins,rRNA, amino acids, tRNAs, GTP, ATP, translation initiation andelongation factors and additional factors associated with translation.Additionally, translation systems may be batch translations orcompartmentalized translation. Batch translation systems combinereaction components in a single compartment while compartmentalizedtranslation systems separate the translation reaction components fromreaction products that can inhibit the translation efficiency. Suchtranslation systems are available commercially.

Further, a coupled transcription/translation system may be used. Coupledtranscription/translation systems allow for both transcription of aninput DNA into a corresponding mRNA, which is in turn translated by thereaction components. An example of a commercially available coupledtranscription/translation is the Rapid Translation System (RTS, RocheInc.). The system includes a mixture containing E. coli lysate forproviding translational components such as ribosomes and translationfactors. Additionally, an RNA polymerase is included for thetranscription of the input DNA into an mRNA template for use intranslation. RTS can use compartmentalization of the reaction componentsby way of a membrane interposed between reaction compartments, includinga supply/waste compartment and a transcription/translation compartment.

Aminoacylation of tRNA may be performed by other agents, including butnot limited to, transferases, polymerases, catalytic antibodies,multi-functional proteins, and the like.

Lu et al. in Mol. Cell. 2001 October; 8(4):759-69 describe a method inwhich a protein is chemically ligated to a synthetic peptide containingunnatural amino acids (expressed protein ligation).

Microinjection techniques have also been use incorporate unnatural aminoacids into proteins. See, e.g., M. W. Nowak, P. C. Kearney, J. R.Sampson, M. E. Saks, C. G. Labarca, S. K. Silverman, W. G. Zhong, J.Thorson, J. N. Abelson, N. Davidson, P. G. Schultz, D. A. Dougherty andH. A. Lester, Science, 268:439 (1995); and, D. A. Dougherty, Curr. Opin.Chem. Biol., 4:645 (2000). A Xenopus oocyte was coinjected with two RNAspecies made in vitro: an mRNA encoding the target protein with a UAGstop codon at the amino acid position of interest and an ambersuppressor tRNA aminoacylated with the desired unnatural amino acid. Thetranslational machinery of the oocyte then inserts the unnatural aminoacid at the position specified by UAG. This method has allowed in vivostructure-function studies of integral membrane proteins, which aregenerally not amenable to in vitro expression systems. Examples includethe incorporation of a fluorescent amino acid into tachykininneurokinin-2 receptor to measure distances by fluorescence resonanceenergy transfer, see, e.g., G. Turcatti, K. Nemeth, M. D. Edgerton, U.Meseth, F. Talabot, M. Peitsch, J. Knowles, H. Vogel and A. Chollet, J.Biol. Chem., 271:19991 (1996); the incorporation of biotinylated aminoacids to identify surface-exposed residues in ion channels, see, e.g.,J. P. Gallivan, H. A. Lester and D. A. Dougherty, Chem. Biol., 4:739(1997); the use of caged tyrosine analogs to monitor conformationalchanges in an ion channel in real time, see, e.g., J. C. Miller, S. K.Silverman, P. M. England, D. A. Dougherty and H. A. Lester, Neuron,20:619 (1998); and, the use of alpha hydroxy amino acids to change ionchannel backbones for probing their gating mechanisms. See, e.g., P. M.England, Y. Zhang, D. A. Dougherty and H. A. Lester, Cell, 96:89 (1999);and, T. Lu, A. Y. Ting, J. Mainland, L. Y. Jan, P. G. Schultz and J.Yang, Nat. Neurosci., 4:239 (2001).

The ability to incorporate unnatural amino acids directly into proteinsin vivo offers a wide variety of advantages including but not limitedto, high yields of mutant proteins, technical ease, the potential tostudy the mutant proteins in cells or possibly in living organisms andthe use of these mutant proteins in therapeutic treatments anddiagnostic uses. The ability to include unnatural amino acids withvarious sizes, acidities, nucleophilicities, hydrophobicities, and otherproperties into proteins can greatly expand our ability to rationallyand systematically manipulate the structures of proteins, both to probeprotein function and create new proteins or organisms with novelproperties.

In one attempt to site-specifically incorporate para-F-Phe, a yeastamber suppressor tRNAPheCUA/phenylalanyl-tRNA synthetase pair was usedin a p-F-Phe resistant, Phe auxotrophic Escherichia coli strain. See,e.g., R. Furter, Protein Sci. 7:419 (1998).

It may also be possible to obtain expression of a hIFN polynucleotide ofthe present invention using a cell-free (in-vitro) translational system.Translation systems may be cellular or cell-free, and may be prokaryoticor eukaryotic. Cellular translation systems include, but are not limitedto, whole cell preparations such as permeabilized cells or cell cultureswherein a desired nucleic acid sequence can be transcribed to mRNA andthe mRNA translated. Cell-free translation systems are commerciallyavailable and many different types and systems are well-known. Examplesof cell-free systems include, but are not limited to, prokaryoticlysates such as Escherichia coli lysates, and eukaryotic lysates such aswheat germ extracts, insect cell lysates, rabbit reticulocyte lysates,rabbit oocyte lysates and human cell lysates. Eukaryotic extracts orlysates may be preferred when the resulting protein is glycosylated,phosphorylated or otherwise modified because many such modifications areonly possible in eukaryotic systems. Some of these extracts and lysatesare available commercially (Promega; Madison, Wis.; Stratagene; LaJolla, Calif.; Amersham; Arlington Heights, Ill.; GIBCO/BRL; GrandIsland, N.Y.). Membranous extracts, such as the canine pancreaticextracts containing microsomal membranes, are also available which areuseful for translating secretory proteins. In these systems, which caninclude either mRNA as a template (in-vitro translation) or DNA as atemplate (combined in-vitro transcription and translation), the in vitrosynthesis is directed by the ribosomes. Considerable effort has beenapplied to the development of cell-free protein expression systems. See,e.g., Kim, D. M. and J. R. Swartz, Biotechnology and Bioengineering, 74:309-316 (2001); Kim, D. M. and J. R. Swartz, Biotechnology Letters, 22,1537-1542, (2000); Kim, D. M., and J. R. Swartz, Biotechnology Progress,16, 385-390, (2000); Kim, D. M., and J. R. Swartz, Biotechnology andBioengineering, 66, 180-188, (1999); and Patnaik, R. and J. R. Swartz,Biotechniques 24, 862-868, (1998); U.S. Pat. No. 6,337,191; U.S. PatentPublication No. 2002/0081660; WO 00/55353; WO 90/05785, which areincorporated by reference herein. Another approach that may be appliedto the expression of hIFN polypeptides comprising a non-naturallyencoded amino acid includes the mRNA-peptide fusion technique. See,e.g., R. Roberts and J. Szostak, Proc. Natl. Acad. Sci. (USA)94:12297-12302 (1997); A. Frankel, et al., Chemistry & Biology10:1043-1050 (2003). In this approach, an mRNA template linked topuromycin is translated into peptide on the ribosome. If one or moretRNA molecules has been modified, non-natural amino acids can beincorporated into the peptide as well. After the last mRNA codon hasbeen read, puromycin captures the C-terminus of the peptide. If theresulting mRNA-peptide conjugate is found to have interesting propertiesin an in vitro assay, its identity can be easily revealed from the mRNAsequence. In this way, one may screen libraries of hIFN polypeptidescomprising one or more non-naturally encoded amino acids to identifypolypeptides having desired properties. More recently, in vitro ribosometranslations with purified components have been reported that permit thesynthesis of peptides substituted with non-naturally encoded aminoacids. See, e.g., A. Forster et al., Proc. Natl. Acad. Sci. (USA)100:6353 (2003).

Reconstituted translation systems may also be used. Mixtures of purifiedtranslation factors have also been used successfully to translate mRNAinto protein as well as combinations of lysates or lysates supplementedwith purified translation factors such as initiation factor-1 (IF-1),IF-2, IF-3 (α or β), elongation factor T (EF-Tu), or terminationfactors. Cell-free systems may also be coupled transcription/translationsystems wherein DNA is introduced to the system, transcribed into mRNAand the mRNA translated as described in Current Protocols in MolecularBiology (F. M. Ausubel et al. editors, Wiley Interscience, 1993), whichis hereby specifically incorporated by reference. RNA transcribed ineukaryotic transcription system may be in the form of heteronuclear RNA(hnRNA) or 5′-end caps (7-methyl guanosine) and 3′-end poly A tailedmature mRNA, which can be an advantage in certain translation systems.For example, capped mRNAs are translated with high efficiency in thereticulocyte lysate system.

IX. Macromolecular Polymers Coupled to hIFN Polypeptides

Various modifications to the non-natural amino acid polypeptidesdescribed herein can be effected using the compositions, methods,techniques and strategies described herein. These modifications includethe incorporation of further functionality onto the non-natural aminoacid component of the polypeptide, including but not limited to, alabel; a dye; a polymer; a water-soluble polymer; a derivative ofpolyethylene glycol; a photocrosslinker; a radionuclide; a cytotoxiccompound; a drug; an affinity label; a photoaffinity label; a reactivecompound; a resin; a second protein or polypeptide or polypeptideanalog; an antibody or antibody fragment; a metal chelator; a cofactor;a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; anantisense polynucleotide; a saccharide; a water-soluble dendrimer; acyclodextrin; an inhibitory ribonucleic acid; a biomaterial; ananoparticle; a spin label; a fluorophore, a metal-containing moiety; aradioactive moiety; a novel functional group; a group that covalently ornoncovalently interacts with other molecules; a photocaged moiety; anactinic radiation excitable moiety; a photoisomerizable moiety; biotin;a derivative of biotin; a biotin analogue; a moiety incorporating aheavy atom; a chemically cleavable group; a photocleavable group; anelongated side chain; a carbon-linked sugar; a redox-active agent; anamino thioacid; a toxic moiety; an isotopically labeled moiety; abiophysical probe; a phosphorescent group; a chemiluminescent group; anelectron dense group; a magnetic group; an intercalating group; achromophore; an energy transfer agent; a biologically active agent; adetectable label; a small molecule; a quantum dot; a nanotransmitter; aradionucleotide; a radiotransmitter; a neutron-capture agent; or anycombination of the above, or any other desirable compound or substance.As an illustrative, non-limiting example of the compositions, methods,techniques and strategies described herein, the following descriptionwill focus on adding macromolecular polymers to the non-natural aminoacid polypeptide with the understanding that the compositions, methods,techniques and strategies described thereto are also applicable (withappropriate modifications, if necessary and for which one of skill inthe art could make with the disclosures herein) to adding otherfunctionalities, including but not limited to those listed above.

A wide variety of macromolecular polymers and other molecules can belinked to hIFN polypeptides of the present invention to modulatebiological properties of the hIFN polypeptide, and/or provide newbiological properties to the hIFN molecule. These macromolecularpolymers can be linked to the hIFN polypeptide via a naturally encodedamino acid, via a non-naturally encoded amino acid, or any functionalsubstituent of a natural or non-natural amino acid, or any substituentor functional group added to a natural or non-natural amino acid. Themolecular weight of the polymer may be of a wide range, including butnot limited to, between about 100 Da and about 100,000 Da or more. Themolecular weight of the polymer may be between about 100 Da and about100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da,55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da,700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In someembodiments, the molecular weight of the polymer is between about 100 Daand 50,000 Da. In some embodiments, the molecular weight of the polymeris between about 100 Da and 40,000 Da. In some embodiments, themolecular weight of the polymer is between about 1,000 Da and 40,000 Da.In some embodiments, the molecular weight of the polymer is betweenabout 5,000 Da and 40,000 Da. In some embodiments, the molecular weightof the polymer is between about 10,000 Da and 40,000 Da. In someembodiments, the molecular weight of the polymer is 30,000 Da. In someembodiments, the molecular weight of the polymer is 40,000 Da.

The present invention provides substantially homogenous preparations ofpolymer:protein conjugates. “Substantially homogenous” as used hereinmeans that polymer:protein conjugate molecules are observed to begreater than half of the total protein. The polymer:protein conjugatehas biological activity and the present “substantially homogenous”PEGylated hIFN polypeptide preparations provided herein are those whichare homogenous enough to display the advantages of a homogenouspreparation, e.g., ease in clinical application in predictability of lotto lot pharmacokinetics.

One may also choose to prepare a mixture of polymer:protein conjugatemolecules, and the advantage provided herein is that one may select theproportion of mono-polymer:protein conjugate to include in the mixture.Thus, if desired, one may prepare a mixture of various proteins withvarious numbers of polymer moieties attached (i.e., di-, tri-, tetra-,etc.) and combine said conjugates with the mono-polymer:proteinconjugate prepared using the methods of the present invention, and havea mixture with a predetermined proportion of mono-polymer:proteinconjugates.

The polymer selected may be water soluble so that the protein to whichit is attached does not precipitate in an aqueous environment, such as aphysiological environment. The polymer may be branched or unbranched.For therapeutic use of the end-product preparation, the polymer will bepharmaceutically acceptable.

Examples of polymers include but are not limited to polyalkyl ethers andalkoxy-capped analogs thereof (e.g., polyoxyethylene glycol,polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogsthereof, especially polyoxyethylene glycol, the latter is also known aspolyethyleneglycol or PEG); polyvinylpyrrolidones; polyvinylalkylethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyloxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkylacrylamides (e.g., polyhydroxypropylmethacrylamide and derivativesthereof); polyhydroxyalkyl acrylates; polysialic acids and analogsthereof; hydrophilic peptide sequences; polysaccharides and theirderivatives, including dextran and dextran derivatives, e.g.,carboxymethyldextran, dextran sulfates, aminodextran; cellulose and itsderivatives, e.g., carboxymethyl cellulose, hydroxyalkyl celluloses;chitin and its derivatives, e.g., chitosan, succinyl chitosan,carboxymethylchitin, carboxymethylchitosan; hyaluronic acid and itsderivatives; starches; alginates; chondroitin sulfate; albumin; pullulanand carboxymethyl pullulan; polyaminoacids and derivatives thereof,e.g., polyglutamic acids, polylysines, polyaspartic acids,polyaspartamides; maleic anhydride copolymers such as: styrene maleicanhydride copolymer, divinylethyl ether maleic anhydride copolymer;polyvinyl alcohols; copolymers thereof; terpolymers thereof; mixturesthereof; and derivatives of the foregoing.

The proportion of polyethylene glycol molecules to protein moleculeswill vary, as will their concentrations in the reaction mixture. Ingeneral, the optimum ratio (in terms of efficiency of reaction in thatthere is minimal excess unreacted protein or polymer) may be determinedby the molecular weight of the polyethylene glycol selected and on thenumber of available reactive groups available. As relates to molecularweight, typically the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.Similarly, branching of the polymer should be taken into account whenoptimizing these parameters. Generally, the higher the molecular weight(or the more branches) the higher the polymer:protein ratio.

As used herein, and when contemplating PEG: hIFN polypeptide conjugates,the term “therapeutically effective amount” refers to an amount whichgives a decrease in viral levels that provides benefit to a patient. Theamount will vary from one individual to another and will depend upon anumber of factors, including the overall physical condition of thepatient and the underlying cause of the condition. The amount of hIFNpolypeptide used for therapy gives an acceptable decrease in virallevel. A therapeutically effective amount of the present compositionsmay be readily ascertained by one of ordinary skill in the art usingpublicly available materials and procedures.

The water soluble polymer may be any structural form including but notlimited to linear, forked or branched. Typically, the water solublepolymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG),but other water soluble polymers can also be employed. By way ofexample, PEG is used to describe certain embodiments of this invention.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods known to those of ordinary skill in the art(Sandier and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3,pages 138-161). The term “PEG” is used broadly to encompass anypolyethylene glycol molecule, without regard to size or to modificationat an end of the PEG, and can be represented as linked to the hIFNpolypeptide by the formula:XO—(CH₂CH₂O)_(n)—CH₂CH₂—Ywhere n is 2 to 10,000 and X is H or a terminal modification, includingbut not limited to, a C₁₋₄ alkyl, a protecting group, or a terminalfunctional group.

In some cases, a PEG used in the invention terminates on one end withhydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). Alternatively,the PEG can terminate with a reactive group, thereby forming abifunctional polymer. Typical reactive groups can include those reactivegroups that are commonly used to react with the functional groups foundin the 20 common amino acids (including but not limited to, maleimidegroups, activated carbonates (including but not limited to,p-nitrophenyl ester), activated esters (including but not limited to,N-hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well asfunctional groups that are inert to the 20 common amino acids but thatreact specifically with complementary functional groups present innon-naturally encoded amino acids (including but not limited to, azidegroups, alkyne groups). It is noted that the other end of the PEG, whichis shown in the above formula by Y, will attach either directly orindirectly to a hIFN polypeptide via a naturally-occurring ornon-naturally encoded amino acid. For instance, Y may be an amide,carbamate or urea linkage to an amine group (including but not limitedto, the epsilon amine of lysine or the N-terminus) of the polypeptide.Alternatively, Y may be a maleimide linkage to a thiol group (includingbut not limited to, the thiol group of cysteine). Alternatively, Y maybe a linkage to a residue not commonly accessible via the 20 commonamino acids. For example, an azide group on the PEG can be reacted withan alkyne group on the hIFN polypeptide to form a Huisgen[3+2]cycloaddition product. Alternatively, an alkyne group on the PEGcan be reacted with an azide group present in a non-naturally encodedamino acid to form a similar product. In some embodiments, a strongnucleophile (including but not limited to, hydrazine, hydrazide,hydroxylamine, semicarbazide) can be reacted with an aldehyde or ketonegroup present in a non-naturally encoded amino acid to form a hydrazone,oxime or semicarbazone, as applicable, which in some cases can befurther reduced by treatment with an appropriate reducing agent.Alternatively, the strong nucleophile can be incorporated into the hIFNpolypeptide via a non-naturally encoded amino acid and used to reactpreferentially with a ketone or aldehyde group present in the watersoluble polymer.

Any molecular mass for a PEG can be used as practically desired,including but not limited to, from about 100 Daltons (Da) to 100,000 Daor more as desired (including but not limited to, sometimes 0.1-50 kDaor 10-40 kDa). The molecular weight of PEG may be of a wide range,including but not limited to, between about 100 Da and about 100,000 Daor more. PEG may be between about 100 Da and about 100,000 Da, includingbut not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da,45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, PEG isbetween about 100 Da and 50,000 Da. In some embodiments, PEG is betweenabout 100 Da and 40,000 Da. In some embodiments, PEG is between about1,000 Da and 40,000 Da. In some embodiments, PEG is between about 5,000Da and 40,000 Da. In some embodiments, PEG is between about 10,000 Daand 40,000 Da. In some embodiments, the molecular weight of the PEG is30,000 Da. In some embodiments, the molecular weight of the PEG is40,000 Da. Branched chain PEGs, including but not limited to, PEGmolecules with each chain having a MW ranging from 1-100 kDa (includingbut not limited to, 1-50 kDa or 5-20 kDa) can also be used. Themolecular weight of each chain of the branched chain PEG may be,including but not limited to, between about 1,000 Da and about 100,000Da or more. The molecular weight of each chain of the branched chain PEGmay be between about 1,000 Da and about 100,000 Da, including but notlimited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da,75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da,10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da,3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecularweight of each chain of the branched chain PEG is between about 1,000 Daand 50,000 Da. In some embodiments, the molecular weight of each chainof the branched chain PEG is between about 1,000 Da and 40,000 Da. Insome embodiments, the molecular weight of each chain of the branchedchain PEG is between about 5,000 Da and 40,000 Da. In some embodiments,the molecular weight of each chain of the branched chain PEG is betweenabout 5,000 Da and 20,000 Da. A wide range of PEG molecules aredescribed in, including but not limited to, the Shearwater Polymers,Inc. catalog, Nektar Therapeutics catalog, incorporated herein byreference.

Generally, at least one terminus of the PEG molecule is available forreaction with the non-naturally-encoded amino acid. For example, PEGderivatives bearing alkyne and azide moieties for reaction with aminoacid side chains can be used to attach PEG to non-naturally encodedamino acids as described herein. If the non-naturally encoded amino acidcomprises an azide, then the PEG will typically contain either an alkynemoiety to effect formation of the [3+2] cycloaddition product or anactivated PEG species (i.e., ester, carbonate) containing a phosphinegroup to effect formation of the amide linkage. Alternatively, if thenon-naturally encoded amino acid comprises an alkyne, then the PEG willtypically contain an azide moiety to effect formation of the [3+2]Huisgen cycloaddition product. If the non-naturally encoded amino acidcomprises a carbonyl group, the PEG will typically comprise a potentnucleophile (including but not limited to, a hydrazide, hydrazine,hydroxylamine, or semicarbazide functionality) in order to effectformation of corresponding hydrazone, oxime, and semicarbazone linkages,respectively. In other alternatives, a reverse of the orientation of thereactive groups described above can be used, i.e., an azide moiety inthe non-naturally encoded amino acid can be reacted with a PEGderivative containing an alkyne.

In some embodiments, the hIFN polypeptide variant with a PEG derivativecontains a chemical functionality that is reactive with the chemicalfunctionality present on the side chain of the non-naturally encodedamino acid.

The invention provides in some embodiments azide- andacetylene-containing polymer derivatives comprising a water solublepolymer backbone having an average molecular weight from about 800 Da toabout 100,000 Da. The polymer backbone of the water-soluble polymer canbe poly(ethylene glycol). However, it should be understood that a widevariety of water soluble polymers including but not limited topoly(ethylene)glycol and other related polymers, including poly(dextran)and poly(propylene glycol), are also suitable for use in the practice ofthis invention and that the use of the term PEG or poly(ethylene glycol)is intended to encompass and include all such molecules. The term PEGincludes, but is not limited to, poly(ethylene glycol) in any of itsforms, including bifunctional PEG, multiarmed PEG, derivatized PEG,forked PEG, branched PEG, pendent PEG (i.e. PEG or related polymershaving one or more functional groups pendent to the polymer backbone),or PEG with degradable linkages therein.

PEG is typically clear, colorless, odorless, soluble in water, stable toheat, inert to many chemical agents, does not hydrolyze or deteriorate,and is generally non-toxic. Poly(ethylene glycol) is considered to bebiocompatible, which is to say that PEG is capable of coexistence withliving tissues or organisms without causing harm. More specifically, PEGis substantially non-immunogenic, which is to say that PEG does not tendto produce an immune response in the body. When attached to a moleculehaving some desirable function in the body, such as a biologicallyactive agent, the PEG tends to mask the agent and can reduce oreliminate any immune response so that an organism can tolerate thepresence of the agent. PEG conjugates tend not to produce a substantialimmune response or cause clotting or other undesirable effects. PEGhaving the formula —CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—, where n is from about3 to about 4000, typically from about 20 to about 2000, is suitable foruse in the present invention. PEG having a molecular weight of fromabout 800 Da to about 100,000 Da are in some embodiments of the presentinvention particularly useful as the polymer backbone. The molecularweight of PEG may be of a wide range, including but not limited to,between about 100 Da and about 100,000 Da or more. The molecular weightof PEG may be between about 100 Da and about 100,000 Da, including butnot limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da,75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da,10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da,3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da,400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecularweight of PEG is between about 100 Da and 50,000 Da. In someembodiments, the molecular weight of PEG is between about 100 Da and40,000 Da. In some embodiments, the molecular weight of PEG is betweenabout 1,000 Da and 40,000 Da. In some embodiments, the molecular weightof PEG is between about 5,000 Da and 40,000 Da. In some embodiments, themolecular weight of PEG is between about 10,000 Da and 40,000 Da. Insome embodiments, the molecular weight of PEG is 30,000 Da. In someembodiments, the molecular weight of PEG is 40,000 Da.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, glycerol oligomers, pentaerythritoland sorbitol. The central branch moiety can also be derived from severalamino acids, such as lysine. The branched poly(ethylene glycol) can berepresented in general form as R(—PEG-OH)_(m) in which R is derived froma core moiety, such as glycerol, glycerol oligomers, or pentaerythritol,and m represents the number of arms. Multi-armed PEG molecules, such asthose described in U.S. Pat. Nos. 5,932,462; 5,643,575; 5,229,490;4,289,872; U.S. Pat. Appl. 2003/0143596; WO 96/21469; and WO 93/21259,each of which is incorporated by reference herein in its entirety, canalso be used as the polymer backbone.

Branched PEG can also be in the form of a forked PEG represented byPEG(—YCHZ₂)_(n), where Y is a linking group and Z is an activatedterminal group linked to CH by a chain of atoms of defined length.

Yet another branched form, the pendant PEG, has reactive groups, such ascarboxyl, along the PEG backbone rather than at the end of PEG chains.

In addition to these forms of PEG, the polymer can also be prepared withweak or degradable linkages in the backbone. For example, PEG can beprepared with ester linkages in the polymer backbone that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:—PEG-CO₂—PEG-+H₂O→PEG-CO₂H+HO-PEG-It is understood by those of ordinary skill in the art that the termpoly(ethylene glycol) or PEG represents or includes all the forms knownin the art including but not limited to those disclosed herein.

Many other polymers are also suitable for use in the present invention.In some embodiments, polymer backbones that are water-soluble, with from2 to about 300 termini, are particularly useful in the invention.Examples of suitable polymers include, but are not limited to, otherpoly(alkylene glycols), such as poly(propylene glycol) (“PPG”),copolymers thereof (including but not limited to copolymers of ethyleneglycol and propylene glycol), terpolymers thereof, mixtures thereof, andthe like. Although the molecular weight of each chain of the polymerbackbone can vary, it is typically in the range of from about 800 Da toabout 100,000 Da, often from about 6,000 Da to about 80,000 Da. Themolecular weight of each chain of the polymer backbone may be betweenabout 100 Da and about 100,000 Da, including but not limited to, 100,000Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da,65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da,8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da,1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200Da, and 100 Da. In some embodiments, the molecular weight of each chainof the polymer backbone is between about 100 Da and 50,000 Da. In someembodiments, the molecular weight of each chain of the polymer backboneis between about 100 Da and 40,000 Da. In some embodiments, themolecular weight of each chain of the polymer backbone is between about1,000 Da and 40,000 Da. In some embodiments, the molecular weight ofeach chain of the polymer backbone is between about 5,000 Da and 40,000Da. In some embodiments, the molecular weight of each chain of thepolymer backbone is between about 10,000 Da and 40,000 Da.

Those of ordinary skill in the art will recognize that the foregoinglist for substantially water soluble backbones is by no means exhaustiveand is merely illustrative, and that all polymeric materials having thequalities described above are contemplated as being suitable for use inthe present invention.

In some embodiments of the present invention the polymer derivatives are“multi-functional”, meaning that the polymer backbone has at least twotermini, and possibly as many as about 300 termini, functionalized oractivated with a functional group. Multifunctional polymer derivativesinclude, but are not limited to, linear polymers having two termini,each terminus being bonded to a functional group which may be the sameor different.

In one embodiment, the polymer derivative has the structure:X-A-POLY-B—N═N═Nwherein:N═N═N is an azide moiety;B is a linking moiety, which may be present or absent;POLY is a water-soluble non-antigenic polymer;A is a linking moiety, which may be present or absent and which may bethe same as B or different; andX is a second functional group.Examples of a linking moiety for A and B include, but are not limitedto, a multiply-functionalized alkyl group containing up to 18, and maycontain between 1-10 carbon atoms. A heteroatom such as nitrogen, oxygenor sulfur may be included with the alkyl chain. The alkyl chain may alsobe branched at a heteroatom. Other examples of a linking moiety for Aand B include, but are not limited to, a multiply functionalized arylgroup, containing up to 10 and may contain 5-6 carbon atoms. The arylgroup may be substituted with one more carbon atoms, nitrogen, oxygen orsulfur atoms. Other examples of suitable linking groups include thoselinking groups described in U.S. Pat. Nos. 5,932,462; 5,643,575; andU.S. Pat. Appl. Publication 2003/0143596, each of which is incorporatedby reference herein. Those of ordinary skill in the art will recognizethat the foregoing list for linking moieties is by no means exhaustiveand is merely illustrative, and that all linking moieties having thequalities described above are contemplated to be suitable for use in thepresent invention.

Examples of suitable functional groups for use as X include, but are notlimited to, hydroxyl, protected hydroxyl, alkoxyl, active ester, such asN-hydroxysuccinimidyl esters and 1-benzotriazolyl esters, activecarbonate, such as N-hydroxysuccinimidyl carbonates and 1-benzotriazolylcarbonates, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate,methacrylate, acrylamide, active sulfone, amine, aminooxy, protectedamine, hydrazide, protected hydrazide, protected thiol, carboxylic acid,protected carboxylic acid, isocyanate, isothiocyanate, maleimide,vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide,glyoxals, diones, mesylates, tosylates, tresylate, alkene, ketone, andazide. As is understood by those of ordinary skill in the art, theselected X moiety should be compatible with the azide group so thatreaction with the azide group does not occur. The azide-containingpolymer derivatives may be homobifunctional, meaning that the secondfunctional group (i.e., X) is also an azide moiety, orheterobifunctional, meaning that the second functional group is adifferent functional group.

The term “protected” refers to the presence of a protecting group ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin the present invention.

Specific examples of terminal functional groups in the literatureinclude, but are not limited to, N-succinimidyl carbonate (see e.g.,U.S. Pat. Nos. 5,281,698, 5,468,478), amine (see, e.g., Buckmann et al.Makromol. Chem. 182:1379 (1981), Zalipsky et al. Eur. Polym. J. 19:1177(1983)), hydrazide (See, e.g., Andresz et al. Makromol. Chem. 179:301(1978)), succinimidyl propionate and succinimidyl butanoate (see, e.g.,Olson et al. in Poly(ethylene glycol) Chemistry & BiologicalApplications, pp 170-181, Harris & Zalipsky Eds., ACS, Washington, D.C.,1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate (See,e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) andJoppich et al. Makromol. Chem. 180:1381 (1979), succinimidyl ester (see,e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see, e.g., U.S.Pat. No. 5,650,234), glycidyl ether (see, e.g., Plitha et al. Eur. J.Biochem. 94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354(1991), oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal.Biochem. 131:25 (1983), Tondelli et al. J. Controlled Release 1:251(1985)), p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl.Biochem. Biotech., 11: 141 (1985); and Sartore et al., Appl. Biochem.Biotech., 27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym.Sci. Chem. Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No.5,252,714), maleimide (see, e.g., Goodson et al. Biotechnology (NY)8:343 (1990), Romani et al. in Chemistry of Peptides and Proteins 2:29(1984)), and Kogan, Synthetic Comm. 22:2417 (1992)),orthopyridyl-disulfide (see, e.g., Woghiren, et al. Bioconj. Chem. 4:314(1993)), acrylol (see, e.g., Sawhney et al., Macromolecules, 26:581(1993)), vinylsulfone (see, e.g., U.S. Pat. No. 5,900,461). All of theabove references and patents are incorporated herein by reference.

In certain embodiments of the present invention, the polymer derivativesof the invention comprise a polymer backbone having the structure:X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—O—(CH₂)_(m)—W—N═N═Nwherein:X is a functional group as described above; andn is about 20 to about 4000.In another embodiment, the polymer derivatives of the invention comprisea polymer backbone having the structure:X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—O—(CH₂)_(m)—W—N═N═Nwherein:W is an aliphatic or aromatic linker moiety comprising between 1-10carbon atoms;n is about 20 to about 4000; andX is a functional group as described above. m is between 1 and 10.

The azide-containing PEG derivatives of the invention can be prepared bya variety of methods known in the art and/or disclosed herein. In onemethod, shown below, a water soluble polymer backbone having an averagemolecular weight from about 800 Da to about 100,000 Da, the polymerbackbone having a first terminus bonded to a first functional group anda second terminus bonded to a suitable leaving group, is reacted with anazide anion (which may be paired with any of a number of suitablecounter-ions, including sodium, potassium, tert-butylammonium and soforth). The leaving group undergoes a nucleophilic displacement and isreplaced by the azide moiety, affording the desired azide-containing PEGpolymer.X—PEG-L+N₃ ⁻→X—PEG-N₃

As shown, a suitable polymer backbone for use in the present inventionhas the formula X—PEG-L, wherein PEG is poly(ethylene glycol) and X is afunctional group which does not react with azide groups and L is asuitable leaving group. Examples of suitable functional groups include,but are not limited to, hydroxyl, protected hydroxyl, acetal, alkenyl,amine, aminooxy, protected amine, protected hydrazide, protected thiol,carboxylic acid, protected carboxylic acid, maleimide, dithiopyridine,and vinylpyridine, and ketone. Examples of suitable leaving groupsinclude, but are not limited to, chloride, bromide, iodide, mesylate,tresylate, and tosylate.

In another method for preparation of the azide-containing polymerderivatives of the present invention, a linking agent bearing an azidefunctionality is contacted with a water soluble polymer backbone havingan average molecular weight from about 800 Da to about 100,000 Da,wherein the linking agent bears a chemical functionality that will reactselectively with a chemical functionality on the PEG polymer, to form anazide-containing polymer derivative product wherein the azide isseparated from the polymer backbone by a linking group.

An exemplary reaction scheme is shown below:X—PEG-M+N-linker-N═N═N→PG-X—PEG-linker-N═N═Nwherein:PEG is poly(ethylene glycol) and X is a capping group such as alkoxy ora functional group as described above; andM is a functional group that is not reactive with the azidefunctionality but that will react efficiently and selectively with the Nfunctional group.

Examples of suitable functional groups include, but are not limited to,M being a carboxylic acid, carbonate or active ester if N is an amine; Mbeing a ketone if N is a hydrazide or aminooxy moiety; M being a leavinggroup if N is a nucleophile.

Purification of the crude product may be accomplished by known methodsincluding, but are not limited to, precipitation of the product followedby chromatography, if necessary.

A more specific example is shown below in the case of PEG diamine, inwhich one of the amines is protected by a protecting group moiety suchas tert-butyl-Boc and the resulting mono-protected PEG diamine isreacted with a linking moiety that bears the azide functionality:BocHN-PEG-NH₂+HO₂C—(CH₂)₃—N═N═N

In this instance, the amine group can be coupled to the carboxylic acidgroup using a variety of activating agents such as thionyl chloride orcarbodiimide reagents and N-hydroxysuccinimide or N-hydroxybenzotriazoleto create an amide bond between the monoamine PEG derivative and theazide-bearing linker moiety. After successful formation of the amidebond, the resulting N-tert-butyl-Boc-protected azide-containingderivative can be used directly to modify bioactive molecules or it canbe further elaborated to install other useful functional groups. Forinstance, the N-t-Boc group can be hydrolyzed by treatment with strongacid to generate an omega-amino-PEG-azide. The resulting amine can beused as a synthetic handle to install other useful functionality such asmaleimide groups, activated disulfides, activated esters and so forthfor the creation of valuable heterobifunctional reagents.

Heterobifunctional derivatives are particularly useful when it isdesired to attach different molecules to each terminus of the polymer.For example, the omega-N-amino-N-azido PEG would allow the attachment ofa molecule having an activated electrophilic group, such as an aldehyde,ketone, activated ester, activated carbonate and so forth, to oneterminus of the PEG and a molecule having an acetylene group to theother terminus of the PEG.

In another embodiment of the invention, the polymer derivative has thestructure:X-A-POLY-B—C≡C—Rwherein:R can be either H or an alkyl, alkene, alkyoxy, or aryl or substitutedaryl group;B is a linking moiety, which may be present or absent;POLY is a water-soluble non-antigenic polymer;A is a linking moiety, which may be present or absent and which may bethe same as B or different; andX is a second functional group.

Examples of a linking moiety for A and B include, but are not limitedto, a multiply-functionalized alkyl group containing up to 18, and maycontain between 1-10 carbon atoms. A heteroatom such as nitrogen, oxygenor sulfur may be included with the alkyl chain. The alkyl chain may alsobe branched at a heteroatom. Other examples of a linking moiety for Aand B include, but are not limited to, a multiply functionalized arylgroup, containing up to 10 and may contain 5-6 carbon atoms. The arylgroup may be substituted with one more carbon atoms, nitrogen, oxygen,or sulfur atoms. Other examples of suitable linking groups include thoselinking groups described in U.S. Pat. Nos. 5,932,462 and 5,643,575 andU.S. Pat. Appl. Publication 2003/0143596, each of which is incorporatedby reference herein. Those of ordinary skill in the art will recognizethat the foregoing list for linking moieties is by no means exhaustiveand is intended to be merely illustrative, and that a wide variety oflinking moieties having the qualities described above are contemplatedto be useful in the present invention.

Examples of suitable functional groups for use as X include hydroxyl,protected hydroxyl, alkoxyl, active ester, such as N-hydroxysuccinimidylesters and 1-benzotriazolyl esters, active carbonate, such asN-hydroxysuccinimidyl carbonates and 1-benzotriazolyl carbonates,acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, ketone, and acetylene. Aswould be understood, the selected X moiety should be compatible with theacetylene group so that reaction with the acetylene group does notoccur. The acetylene-containing polymer derivatives may behomobifunctional, meaning that the second functional group (i.e., X) isalso an acetylene moiety, or heterobifunctional, meaning that the secondfunctional group is a different functional group.

In another embodiment of the present invention, the polymer derivativescomprise a polymer backbone having the structure:X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—O—(CH₂)_(m)—C≡CHwherein:X is a functional group as described above;n is about 20 to about 4000; andm is between 1 and 10.Specific examples of each of the heterobifunctional PEG polymers areshown below.

The acetylene-containing PEG derivatives of the invention can beprepared using methods known to those of ordinary skill in the artand/or disclosed herein. In one method, a water soluble polymer backbonehaving an average molecular weight from about 800 Da to about 100,000Da, the polymer backbone having a first terminus bonded to a firstfunctional group and a second terminus bonded to a suitable nucleophilicgroup, is reacted with a compound that bears both an acetylenefunctionality and a leaving group that is suitable for reaction with thenucleophilic group on the PEG. When the PEG polymer bearing thenucleophilic moiety and the molecule bearing the leaving group arecombined, the leaving group undergoes a nucleophilic displacement and isreplaced by the nucleophilic moiety, affording the desiredacetylene-containing polymer.X—PEG-Nu+L-A-C→X—PEG-Nu-A-C≡CR′

As shown, a preferred polymer backbone for use in the reaction has theformula X—PEG-Nu, wherein PEG is poly(ethylene glycol), Nu is anucleophilic moiety and X is a functional group that does not react withNu, L or the acetylene functionality.

Examples of Nu include, but are not limited to, amine, alkoxy, aryloxy,sulfhydryl, imino, carboxylate, hydrazide, aminoxy groups that wouldreact primarily via a SN2-type mechanism. Additional examples of Nugroups include those functional groups that would react primarily via annucleophilic addition reaction. Examples of L groups include chloride,bromide, iodide, mesylate, tresylate, and tosylate and other groupsexpected to undergo nucleophilic displacement as well as ketones,aldehydes, thioesters, olefins, alpha-beta unsaturated carbonyl groups,carbonates and other electrophilic groups expected to undergo additionby nucleophiles.

In another embodiment of the present invention, A is an aliphatic linkerof between 1-10 carbon atoms or a substituted aryl ring of between 6-14carbon atoms. X is a functional group which does not react with azidegroups and L is a suitable leaving group

In another method for preparation of the acetylene-containing polymerderivatives of the invention, a PEG polymer having an average molecularweight from about 800 Da to about 100,000 Da, bearing either a protectedfunctional group or a capping agent at one terminus and a suitableleaving group at the other terminus is contacted by an acetylene anion.

An exemplary reaction scheme is shown below:X—PEG-L+—C≡CR′→X—PEG-C≡CR′wherein:PEG is poly(ethylene glycol) and X is a capping group such as alkoxy ora functional group as described above; andR′ is either H, an alkyl, alkoxy, aryl or aryloxy group or a substitutedalkyl, alkoxyl, aryl or aryloxy group.

In the example above, the leaving group L should be sufficientlyreactive to undergo SN2-type displacement when contacted with asufficient concentration of the acetylene anion. The reaction conditionsrequired to accomplish SN2 displacement of leaving groups by acetyleneanions are known to those of ordinary skill in the art.

Purification of the crude product can usually be accomplished by methodsknown in the art including, but are not limited to, precipitation of theproduct followed by chromatography, if necessary.

Water soluble polymers can be linked to the hIFN polypeptides of theinvention. The water soluble polymers may be linked via a non-naturallyencoded amino acid incorporated in the hIFN polypeptide or anyfunctional group or substituent of a non-naturally encoded or naturallyencoded amino acid, or any functional group or substituent added to anon-naturally encoded or naturally encoded amino acid. Alternatively,the water soluble polymers are linked to a hIFN polypeptideincorporating a non-naturally encoded amino acid via anaturally-occurring amino acid (including but not limited to, cysteine,lysine or the amine group of the N-terminal residue). In some cases, thehIFN polypeptides of the invention comprise 1, 2, 3, 4, 5, 6, 7, 8, 9,10 non-natural amino acids, wherein one or more non-naturally-encodedamino acid(s) are linked to water soluble polymer(s) (including but notlimited to, PEG and/or oligosaccharides). In some cases, the hIFNpolypeptides of the invention further comprise 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more naturally-encoded amino acid(s) linked to water solublepolymers. In some cases, the hIFN polypeptides of the invention compriseone or more non-naturally encoded amino acid(s) linked to water solublepolymers and one or more naturally-occurring amino acids linked to watersoluble polymers. In some embodiments, the water soluble polymers usedin the present invention enhance the serum half-life of the hIFNpolypeptide relative to the unconjugated form.

The number of water soluble polymers linked to a hIFN polypeptide (i.e.,the extent of PEGylation or glycosylation) of the present invention canbe adjusted to provide an altered (including but not limited to,increased or decreased) pharmacologic, pharmacokinetic orpharmacodynamic characteristic such as in vivo half-life. In someembodiments, the half-life of hIFN is increased at least about 10, 20,30, 40, 50, 60, 70, 80, 90 percent, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold,14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold,30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or at least about 100-foldover an unmodified polypeptide.

PEG Derivatives Containing a Strong Nucleophilic Group (i.e., Hydrazide,Hydrazine, Hydroxylamine or Semicarbazide)

In one embodiment of the present invention, a hIFN polypeptidecomprising a carbonyl-containing non-naturally encoded amino acid ismodified with a PEG derivative that contains a terminal hydrazine,hydroxylamine, hydrazide or semicarbazide moiety that is linked directlyto the PEG backbone.

In some embodiments, the hydroxylamine-terminal PEG derivative will havethe structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—O—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In some embodiments, the hydrazine- or hydrazide-containing PEGderivative will have the structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—X—NH—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 and X is optionally a carbonyl group (C═O) that can bepresent or absent.

In some embodiments, the semicarbazide-containing PEG derivative willhave the structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—NH—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000.

In another embodiment of the invention, a hIFN polypeptide comprising acarbonyl-containing amino acid is modified with a PEG derivative thatcontains a terminal hydroxylamine, hydrazide, hydrazine, orsemicarbazide moiety that is linked to the PEG backbone by means of anamide linkage.

In some embodiments, the hydroxylamine-terminal PEG derivatives have thestructure:RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—O—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In some embodiments, the hydrazine- or hydrazide-containing PEGderivatives have the structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—X—NH—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, n is100-1,000 and X is optionally a carbonyl group (C═O) that can be presentor absent.

In some embodiments, the semicarbazide-containing PEG derivatives havethe structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—NH—C(O)—NH—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000.

In another embodiment of the invention, a hIFN polypeptide comprising acarbonyl-containing amino acid is modified with a branched PEGderivative that contains a terminal hydrazine, hydroxylamine, hydrazideor semicarbazide moiety, with each chain of the branched PEG having a MWranging from 10-40 kDa and, may be from 5-20 kDa.

In another embodiment of the invention, a hIFN polypeptide comprising anon-naturally encoded amino acid is modified with a PEG derivativehaving a branched structure. For instance, in some embodiments, thehydrazine- or hydrazide-terminal PEG derivative will have the followingstructure:[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—NH—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000, and X is optionally a carbonyl group (C═O) that can bepresent or absent.

In some embodiments, the PEG derivatives containing a semicarbazidegroup will have the structure:[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—C(O)—NH—CH₂—CH₂]₂CH—X—(CH₂)_(m)—NH—C(O)—NH—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000.

In some embodiments, the PEG derivatives containing a hydroxylaminegroup will have the structure:[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—C(O)—NH—CH₂—CH₂]₂CH—X—(CH₂)_(m)—O—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000.

The degree and sites at which the water soluble polymer(s) are linked tothe hIFN polypeptide can modulate the binding of the hIFN polypeptide tothe hIFN polypeptide receptor at Site 1. In some embodiments, thelinkages are arranged such that the hIFN polypeptide binds the hIFNpolypeptide receptor at Site 1 with a K_(d) of about 400 nM or lower,with a K_(d) of 150 nM or lower, and in some cases with a K_(d) of 100nM or lower, as measured by an equilibrium binding assay, such as thatdescribed in Spencer et al., J. Biol. Chem., 263:7862-7867 (1988) forhGH.

Methods and chemistry for activation of polymers as well as forconjugation of peptides are described in the literature and are known inthe art. Commonly used methods for activation of polymers include, butare not limited to, activation of functional groups with cyanogenbromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin,divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine, etc.(see, R. F. Taylor, (1991), PROTEIN IMMOBILISATION. FUNDAMENTAL ANDAPPLICATIONS, Marcel Dekker, N.Y.; S. S. Wong, (1992), CHEMISTRY OFPROTEIN CONJUGATION AND CROSSLINKING, CRC Press, Boca Raton; G. T.Hermanson et al., (1993), IMMOBILIZED AFFINITY LIGAND TECHNIQUES,Academic Press, N.Y.; Dunn, R. L., et al., Eds. POLYMERIC DRUGS AND DRUGDELIVERY SYSTEMS, ACS Symposium Series Vol. 469, American ChemicalSociety, Washington, D.C. 1991).

Several reviews and monographs on the functionalization and conjugationof PEG are available. See, for example, Harris, Macromol. Chem. Phys.C25: 325-373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987);Wong et al., Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al.,Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992);Zalipsky, Bioconjugate Chem. 6: 150-165 (1995).

Methods for activation of polymers can also be found in WO 94/17039,U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No.5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No.5,281,698, and WO 93/15189, and for conjugation between activatedpolymers and enzymes including but not limited to Coagulation FactorVIII (WO 94/15625), hemoglobin (WO 94/09027), oxygen carrying molecule(U.S. Pat. No. 4,412,989), ribonuclease and superoxide dismutase(Veronese at al., App. Biochem. Biotech. 11: 141-52 (1985)). Allreferences and patents cited are incorporated by reference herein.

PEGylation (i.e., addition of any water soluble polymer) of hIFNpolypeptides containing a non-naturally encoded amino acid, such asp-azido-L-phenylalanine, is carried out by any convenient method. Forexample, hIFN polypeptide is PEGylated with an alkyne-terminated mPEGderivative. Briefly, an excess of solid mPEG(5000)-O—CH₂—C≡CH is added,with stirring, to an aqueous solution of p-azido-L-Phe-containing hIFNpolypeptide at room temperature. Typically, the aqueous solution isbuffered with a buffer having a pK_(a) near the pH at which the reactionis to be carried out (generally about pH 4-10). Examples of suitablebuffers for PEGylation at pH 7.5, for instance, include, but are notlimited to, HEPES, phosphate, borate, TRIS-HCl, EPPS, and TES. The pH iscontinuously monitored and adjusted if necessary. The reaction istypically allowed to continue for between about 1-48 hours.

The reaction products are subsequently subjected to hydrophobicinteraction chromatography to separate the PEGylated hIFN polypeptidevariants from free mPEG(5000)-O—CH₂—C≡CH and any high-molecular weightcomplexes of the pegylated hIFN polypeptide which may form whenunblocked PEG is activated at both ends of the molecule, therebycrosslinking hIFN polypeptide variant molecules. The conditions duringhydrophobic interaction chromatography are such that freemPEG(5000)-O—CH₂—C≡CH flows through the column, while any crosslinkedPEGylated hIFN polypeptide variant complexes elute after the desiredforms, which contain one hIFN polypeptide variant molecule conjugated toone or more PEG groups. Suitable conditions vary depending on therelative sizes of the cross-linked complexes versus the desiredconjugates and are readily determined by those of ordinary skill in theart. The eluent containing the desired conjugates is concentrated byultrafiltration and desalted by diafiltration.

If necessary, the PEGylated hIFN polypeptide obtained from thehydrophobic chromatography can be purified further by one or moreprocedures known to those of ordinary skill in the art including, butare not limited to, affinity chromatography; anion- or cation-exchangechromatography (using, including but not limited to, DEAE SEPHAROSE);chromatography on silica; reverse phase HPLC; gel filtration (using,including but not limited to, SEPHADEX G-75); hydrophobic interactionchromatography; size-exclusion chromatography, metal-chelatechromatography; ultrafiltration/diafiltration; ethanol precipitation;ammonium sulfate precipitation; chromatofocusing; displacementchromatography; electrophoretic procedures (including but not limited topreparative isoelectric focusing), differential solubility (includingbut not limited to ammonium sulfate precipitation), or extraction.Apparent molecular weight may be estimated by GPC by comparison toglobular protein standards (Preneta, A Z in PROTEIN PURIFICATIONMETHODS, A PRACTICAL APPROACH (Harris & Angal, Eds.) IRL Press 1989,293-306). The purity of the hGH-PEG conjugate can be assessed byproteolytic degradation (including but not limited to, trypsin cleavage)followed by mass spectrometry analysis. Pepinsky R B., et al., J.Pharmcol. & Exp. Ther. 297(3):1059-66 (2001).

A water soluble polymer linked to an amino acid of a hIFN polypeptide ofthe invention can be further derivatized or substituted withoutlimitation.

Azide-Containing PEG Derivatives

In another embodiment of the invention, a hIFN polypeptide is modifiedwith a PEG derivative that contains an azide moiety that will react withan alkyne moiety present on the side chain of the non-naturally encodedamino acid. In general, the PEG derivatives will have an averagemolecular weight ranging from 1-100 kDa and, in some embodiments, from10-40 kDa.

In some embodiments, the azide-terminal PEG derivative will have thestructure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—N₃where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In another embodiment, the azide-terminal PEG derivative will have thestructure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—(CH₂)_(p)—N₃where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10 and n is 100-1,000 (i.e., average molecular weight is between 5-40kDa).

In another embodiment of the invention, a hIFN polypeptide comprising aalkyne-containing amino acid is modified with a branched PEG derivativethat contains a terminal azide moiety, with each chain of the branchedPEG having a MW ranging from 10-40 kDa and may be from 5-20 kDa. Forinstance, in some embodiments, the azide-terminal PEG derivative willhave the following structure:[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—(CH₂)_(p)N₃where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10, and n is 100-1,000, and X is optionally an O, N, S or carbonylgroup (C═O), in each case that can be present or absent.Alkyne-Containing PEG Derivatives

In another embodiment of the invention, a hIFN polypeptide is modifiedwith a PEG derivative that contains an alkyne moiety that will reactwith an azide moiety present on the side chain of the non-naturallyencoded amino acid.

In some embodiments, the alkyne-terminal PEG derivative will have thefollowing structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—C≡CHwhere R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In another embodiment of the invention, a hIFN polypeptide comprising analkyne-containing non-naturally encoded amino acid is modified with aPEG derivative that contains a terminal azide or terminal alkyne moietythat is linked to the PEG backbone by means of an amide linkage.

In some embodiments, the alkyne-terminal PEG derivative will have thefollowing structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—(CH₂)_(p)—C≡CHwhere R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10 and n is 100-1,000.

In another embodiment of the invention, a hIFN polypeptide comprising anazide-containing amino acid is modified with a branched PEG derivativethat contains a terminal alkyne moiety, with each chain of the branchedPEG having a MW ranging from 10-40 kDa and may be from 5-20 kDa. Forinstance, in some embodiments, the alkyne-terminal PEG derivative willhave the following structure:[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—(CH₂)_(p)C≡CHwhere R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10, and n is 100-1,000, and X is optionally an O, N, S or carbonylgroup (C═O), or not present.Phosphine-Containing PEG Derivatives

In another embodiment of the invention, a hIFN polypeptide is modifiedwith a PEG derivative that contains an activated functional group(including but not limited to, ester, carbonate) further comprising anaryl phosphine group that will react with an azide moiety present on theside chain of the non-naturally encoded amino acid. In general, the PEGderivatives will have an average molecular weight ranging from 1-100 kDaand, in some embodiments, from 10-40 kDa.

In some embodiments, the PEG derivative will have the structure:

wherein n is 1-10; X can be O, N, S or not present, Ph is phenyl, and Wis a water soluble polymer.

In some embodiments, the PEG derivative will have the structure:

wherein X can be O, N, S or not present, Ph is phenyl, W is a watersoluble polymer and R can be H, alkyl, aryl, substituted alkyl andsubstituted aryl groups. Exemplary R groups include but are not limitedto —CH₂, —C(CH₃)₃, —OR′, —NR′R″, —SR, -halogen, —C(O)R′, —CONR′R″,—S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂. R′, R″, R′″ and R′″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R′″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).Other PEG Derivatives and General PEGylation Techniques

Other exemplary PEG molecules that may be linked to hIFN polypeptides,as well as PEGylation methods include those described in, e.g., U.S.Patent Publication No. 2004/0001838; 2002/0052009; 2003/0162949;2004/0013637; 2003/0228274; 2003/0220447; 2003/0158333; 2003/0143596;2003/0114647; 2003/0105275; 2003/0105224; 2003/0023023; 2002/0156047;2002/0099133; 2002/0086939; 2002/0082345; 2002/0072573; 2002/0052430;2002/0040076; 2002/0037949; 2002/0002250; 2001/0056171; 2001/0044526;2001/0021763; U.S. Pat. Nos. 6,646,110; 5,824,778; 5,476,653; 5,219,564;5,629,384; 5,736,625; 4,902,502; 5,281,698; 5,122,614; 5,473,034;5,516,673; 5,382,657; 6,552,167; 6,610,281; 6,515,100; 6,461,603;6,436,386; 6,214,966; 5,990,237; 5,900,461; 5,739,208; 5,672,662;5,446,090; 5,808,096; 5,612,460; 5,324,844; 5,252,714; 6,420,339;6,201,072; 6,451,346; 6,306,821; 5,559,213; 5,747,646; 5,834,594;5,849,860; 5,980,948; 6,004,573; 6,129,912; WO 97/32607, EP 229,108, EP402,378, WO 92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO94/18247, WO 94/28024, WO 95/00162, WO 95/11924, WO95/13090, WO95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO95/06058, EP 439 508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131,WO 98/05363, EP 809 996, WO 96/41813, WO 96/07670, EP 605 963, EP 510356, EP 400 472, EP 183 503 and EP 154 316, which are incorporated byreference herein. Any of the PEG molecules described herein may be usedin any form, including but not limited to, single chain, branched chain,multiarm chain, single functional, bi-functional, multi-functional, orany combination thereof.

Enhancing Affinity for Serum Albumin

Various molecules can also be fused to the hIFN polypeptides of theinvention to modulate the half-life of hIFN polypeptides in serum. Insome embodiments, molecules are linked or fused to hIFN polypeptides ofthe invention to enhance affinity for endogenous serum albumin in ananimal.

For example, in some cases, a recombinant fusion of a hIFN polypeptideand an albumin binding sequence is made. Exemplary albumin bindingsequences include, but are not limited to, the albumin binding domainfrom streptococcal protein G (see. e.g., Makrides et al., J. Pharmacol.Exp. Ther. 277:534-542 (1996) and Sjolander et al., J. Immunol. Methods201:115-123 (1997)), or albumin-binding peptides such as those describedin, e.g., Dennis, et al., J. Biol. Chem. 277:35035-35043 (2002).

In other embodiments, the hIFN polypeptides of the present invention areacylated with fatty acids. In some cases, the fatty acids promotebinding to serum albumin. See, e.g., Kurtzhals, et al., Biochem. J.312:725-731 (1995).

In other embodiments, the hIFN polypeptides of the invention are fuseddirectly with serum albumin (including but not limited to, human serumalbumin). Those of skill in the art will recognize that a wide varietyof other molecules can also be linked to hIFN in the present inventionto modulate binding to serum albumin or other serum components.

X. Glycosylation of hIFN Polypeptides

The invention includes hIFN polypeptides incorporating one or morenon-naturally encoded amino acids bearing saccharide residues. Thesaccharide residues may be either natural (including but not limited to,N-acetylglucosamine) or non-natural (including but not limited to,3-fluorogalactose). The saccharides may be linked to the non-naturallyencoded amino acids either by an N- or O-linked glycosidic linkage(including but not limited to, N-acetylgalactose-L-serine) or anon-natural linkage (including but not limited to, an oxime or thecorresponding C- or S-linked glycoside).

The saccharide (including but not limited to, glycosyl) moieties can beadded to hIFN polypeptides either in vivo or in vitro. In someembodiments of the invention, a hIFN polypeptide comprising acarbonyl-containing non-naturally encoded amino acid is modified with asaccharide derivatized with an aminooxy group to generate thecorresponding glycosylated polypeptide linked via an oxime linkage. Onceattached to the non-naturally encoded amino acid, the saccharide may befurther elaborated by treatment with glycosyltransferases and otherenzymes to generate an oligosaccharide bound to the hIFN polypeptide.See, e.g., H. Liu, et al. J. Am. Chem. Soc. 125: 1702-1703 (2003).

In some embodiments of the invention, a hIFN polypeptide comprising acarbonyl-containing non-naturally encoded amino acid is modifieddirectly with a glycan with defined structure prepared as an aminooxyderivative. One of ordinary skill in the art will recognize that otherfunctionalities, including azide, alkyne, hydrazide, hydrazine, andsemicarbazide, can be used to link the saccharide to the non-naturallyencoded amino acid.

In some embodiments of the invention, a hIFN polypeptide comprising anazide or alkynyl-containing non-naturally encoded amino acid can then bemodified by, including but not limited to, a Huisgen [3+2]cycloadditionreaction with, including but not limited to, alkynyl or azidederivatives, respectively. This method allows for proteins to bemodified with extremely high selectivity.

XI. GH Supergene Family Member Dimers and Multimers

The present invention also provides for GH supergene family membercombinations (including but not limited to hIFN and hIFN analogs) suchas homodimers, heterodimers, homomultimers, or heteromultimers (i.e.,trimers, tetramers, etc.) where a GH supergene family member polypeptidesuch as hIFN containing one or more non-naturally encoded amino acids isbound to another GH supergene family member or variant thereof or anyother polypeptide that is a non-GH supergene family member or variantthereof, either directly to the polypeptide backbone or via a linker.Due to its increased molecular weight compared to monomers, the GHsupergene family member, such as hIFN, dimer or multimer conjugates mayexhibit new or desirable properties, including but not limited todifferent pharmacological, pharmacokinetic, pharmacodynamic, modulatedtherapeutic half-life, or modulated plasma half-life relative to themonomeric GH supergene family member. In some embodiments, the GHsupergene family member, such as hIFN, dimers of the invention willmodulate the dimerization of the GH supergene family member receptor. Inother embodiments, the GH supergene family member dimers or multimers ofthe present invention will act as a GH supergene family member receptorantagonist, agonist, or modulator.

In some embodiments, one or more of the hIFN molecules present in a hIFNcontaining dimer or multimer comprises a non-naturally encoded aminoacid linked to a water soluble polymer that is present within the SiteII binding region. As such, each of the hIFN molecules of the dimer ormultimer are accessible for binding to the hIFN polypeptide receptor viathe Site I interface but are unavailable for binding to a second hIFNpolypeptide receptor via the Site II interface. Thus, the hIFNpolypeptide dimer or multimer can engage the Site I binding sites ofeach of two distinct hIFN polypeptide receptors but, as the hIFNmolecules have a water soluble polymer attached to a non-geneticallyencoded amino acid present in the Site II region, the hIFN polypeptidereceptors cannot engage the Site II region of the hIFN polypeptideligand and the dimer or multimer acts as a hIFN polypeptide antagonist.In some embodiments, one or more of the hIFN molecules present in a hIFNpolypeptide containing dimer or multimer comprises a non-naturallyencoded amino acid linked to a water soluble polymer that is presentwithin the Site I binding region, allowing binding to the Site IIregion. Alternatively, in some embodiments one or more of the hIFNmolecules present in a hIFN polypeptide containing dimer or multimercomprises a non-naturally encoded amino acid linked to a water solublepolymer that is present at a site that is not within the Site I or SiteII binding region, such that both are available for binding. In someembodiments a combination of hIFN molecules is used having Site I, SiteII, or both available for binding. A combination of hIFN moleculeswherein at least one has Site I available for binding, and at least onehas Site II available for binding may provide molecules having a desiredactivity or property. In addition, a combination of hIFN moleculeshaving both Site I and Site II available for binding may produce asuper-agonist hIFN molecule.

In some embodiments, the GH supergene family member polypeptides arelinked directly, including but not limited to, via an Asn-Lys amidelinkage or Cys-Cys disulfide linkage. In some embodiments, the linked GHsupergene family member polypeptides, and/or the linked non-GH supergenefamily member, will comprise different non-naturally encoded amino acidsto facilitate dimerization, including but not limited to, an alkyne inone non-naturally encoded amino acid of a first hIFN polypeptide and anazide in a second non-naturally encoded amino acid of a second GHsupergene family member polypeptide will be conjugated via a Huisgen[3+2]cycloaddition. Alternatively, a first GH supergene family member,and/or the linked non-GH supergene family member, polypeptide comprisinga ketone-containing non-naturally encoded amino acid can be conjugatedto a second GH supergene family member polypeptide comprising ahydroxylamine-containing non-naturally encoded amino acid and thepolypeptides are reacted via formation of the corresponding oxime.

Alternatively, the two GH supergene family member polypeptides, and/orthe linked non-GH supergene family member, are linked via a linker. Anyhetero- or homo-bifunctional linker can be used to link the two GHsupergene family members, and/or the linked non-GH supergene familymember, polypeptides, which can have the same or different primarysequence. In some cases, the linker used to tether the GH supergenefamily member, and/or the linked non-GH supergene family member,polypeptides together can be a bifunctional PEG reagent. The linker mayhave a wide range of molecular weight or molecular length. Larger orsmaller molecular weight linkers may be used to provide a desiredspatial relationship or conformation between the GH superfamily memberand the linked entity. Linkers having longer or shorter molecular lengthmay also be used to provide a desired space or flexibility between theGH superfamily member and the linked entity. Similarly, a linker havinga particular shape or conformation may be utilized to impart aparticular shape or conformation to the GH superfamily member or thelinked entity, either before or after the GH superfamily member reachesits target. This optimization of the spatial relationship between the GHsuperfamily member and the linked entity may provide new, modulated, ordesired properties to the molecule.

In some embodiments, the invention provides water-soluble bifunctionallinkers that have a dumbbell structure that includes: a) an azide, analkyne, a hydrazine, a hydrazide, a hydroxylamine, or acarbonyl-containing moiety on at least a first end of a polymerbackbone; and b) at least a second functional group on a second end ofthe polymer backbone. The second functional group can be the same ordifferent as the first functional group. The second functional group, insome embodiments, is not reactive with the first functional group. Theinvention provides, in some embodiments, water-soluble compounds thatcomprise at least one arm of a branched molecular structure. Forexample, the branched molecular structure can be dendritic.

In some embodiments, the invention provides multimers comprising one ormore GH supergene family member, such as hIFN, formed by reactions withwater soluble activated polymers that have the structure:R—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—Xwherein n is from about 5 to 3,000, m is 2-10, X can be an azide, analkyne, a hydrazine, a hydrazide, an aminooxy group, a hydroxylamine, anacetyl, or carbonyl-containing moiety, and R is a capping group, afunctional group, or a leaving group that can be the same or differentas X. R can be, for example, a functional group selected from the groupconsisting of hydroxyl, protected hydroxyl, alkoxyl,N-hydroxysuccinimidyl ester, 1-benzotriazolyl ester,N-hydroxysuccinimidyl carbonate, 1-benzotriazolyl carbonate, acetal,aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, and ketone.XII. Measurement of hIFN Polypeptide Activity and Affinity of hIFNPolypeptide for the hIFN Polypeptide Receptor

The hIFN receptor can be prepared as described in U.S. Pat. No.6,566,132; 5,889,151; 5,861,258; 5,731,169; 5,578,707, which isincorporated by reference herein. hIFN polypeptide activity can bedetermined using standard or known in vitro or in vivo assays. Forexample, cells or cell lines that modulate growth or MHC Class I or IIantigen production in response to hIFN or bind hIFN (including but notlimited to, cells containing active IFN receptors such as humanlymphoblastoid Daudi cells, or recombinant IFN receptor producing cells)can be used to monitor hIFN receptor binding. For a non-PEGylated orPEGylated hIFN polypeptide comprising a non-natural amino acid, theaffinity of the hormone for its receptor can be measured by usingtechniques known in the art such as a BIAcore™ biosensor (Pharmacia). Invivo animal models as well as human clinical trials for testing hIFNactivity include those described in, e.g., Kontsek et al., Acta Virol.43:63 (1999); Youngster et al., Current Pharma Design 8:2139 (2002);Kozlowski et al., BioDrugs 15:419 (2001); U.S. Pat. No. 6,180,096;6,177,074; 6,042,822; 5,981,709; 5,951,974; 5,908,621; 5,711,944;5,738,846, which are incorporated by reference herein.

Regardless of which methods are used to create the present hIFN analogs,the analogs are subject to assays for biological activity. Tritiatedthymidine assays may be conducted to ascertain the degree of celldivision. Other biological assays, however, may be used to ascertain thedesired activity. Biological assays such as assaying for the ability toinhibit viral replication, also provides indication of IFN activity. SeeBailon et al., Bioconj. Chem. 12:195 (2001); Forti et al., Meth.Enzymol. 119:533 (1986); Walter et al., Cancer Biother. & Radiopharm.13:143 (1998); DiMarco et al., BioChem. Biophys. Res. Com. 202:1445(1994); Foser et al. Pharmacogenomics Journal 3:312-319 (2003); and U.S.Pat. No. 4,675,282; 4,241,174; 4,514,507; 4,622,292; 5,766,864, whichare incorporated by reference herein. Assays such as those described byOritani et al. Nature Medicine 2000 6(6):659-666; Kawamoto et al.Experimental Hematology 2004 32:797-805; and Kawamoto et al. J. Virol.2003 September; 77(17):9622-31 may be also used to assess the biologicalactivity and potential side effects of hIFN polypeptides of theinvention.

Platanias et al. in Experimental Hematology 1999; 27:1583-1592, which isincorporated by reference herein, discuss signaling pathways activatedby interferons including the Jak-Stat pathway, the insulin receptorsubstrate (IRS)/PI-3′-kinase pathway, and the activation of CBL and theCrk-signaling pathway. The importance of the Vav proto-oncogene and itsrole in IFN-dependent growth inhibition and the role of tyrosinephosphatases in type I IFN signaling, as well as molecules critical tosignaling cascades with the type II interferon receptor were discussed.Assays evaluating signaling and pathways downstream from interferonreceptor binding may be used to evaluate hIFN polypeptides of theinvention, including studies described in Platanias et al. inExperimental Hematology 1999 27:1315-1321, which is incorporated byreference herein, that investigate two members of the Crk-family ofproteins, CrkL and CrkII.

To assess the effect of hIFN polypeptides on normal myeloid (CFU-GM) anderythroid progenitors (BFU-E), colony formation assays such as thosedescribed by Kawamoto et al. Experimental Hematology 2004 32:797-805 orGiron-Michel, Leukemia 2002 16:1135-1142, incorporated by referenceherein, may be used. Clonal proliferation of megakaryocytes may also beperformed. There exists a correlation between colony formation assaysand bone marrow toxicity. In vivo effects on normal hematopoiesis may bemeasured in, for example, peripheral blood or bone marrow of mice afterinjection with hIFN polypeptides or effects on body temperature in micemay be measured as described by Kawamoto et al. Oligoadenylatesynthetase mRNA may also be used as a biomarker for antiviral activity(branched DNA assay).

Viral replication assays or in vivo studies may be performed with hIFNpolypeptides of the invention to screen for anti-viral activity. Viralreplication assays are known to those skilled in the art and may involveHCV (Hepatitis C Virus), VSV (Vesicular Stomatitis Virus), or EMCV(Encephalomyocarditis Virus). HCV replicon assays involving human WISHor Huh-7 cells may be performed with RNA expression measured by variousmethods including, but not limited to, RT-PCR, Real Time PCR, orbranched DNA methods. Different HCV genotypes may be used, including butnot limited to, HCV genotypes 1a and 1b. The reduction of cytopathiceffect (CPE) of cells such as baby hamster kidney BHK21 cells infectedwith VSV may also be measured with hIFN polypeptides. Various cell linesand calculation algorithms may be used to determine potency in CPEassays. Comparisons are made with known reference standards andinternational units may be calculated. Other assays include, but are notlimited to, assays that involve HCV replication in specific cell linesand human foreskin fibroblast cells infected with EMCV (Trotta et al.,Drug Information Journal (2000); 34:1231-1246), which is incorporated byreference herein.

Other in vitro assays may be used to ascertain biological activity. Ingeneral, the test for biological activity should provide analysis forthe desired result, such as increase or decrease in biological activity(as compared to non-altered IFN), different biological activity (ascompared to non-altered IFN), receptor affinity analysis, or serumhalf-life analysis.

It was previously reported that Daudi cells will bind ¹²⁵I-labeledmurine IFN and that this binding can be competed for by addition ofunlabeled IFN (See e.g. U.S. Pat. Nos. 5,516,515; 5,632,988). Theability of natural IFN and hIFN to compete for binding of ¹²⁵I-IFN tohuman and murine leukemic cells is tested. Highly purified natural IFN(>95% pure; 1 μg) is iodinated [Tejedor, et al., Anal. Biochem., 127,143 (1982)], and is separated from reactants by gel filtration and ionexchange chromatography. The specific activity of the natural ¹²⁵I-IFNmay be approximately 100 μCi/μg protein.

Trotta et al. Drug Information Journal 2000; 34:1231-1246, which isincorporated by reference herein, discuss the numerous biologicalactivities of IFNα including immunomodulatory, antiproliferative,antiviral, and antimicrobial activities. Different human IFN a speciesexhibit varied relative levels of biological activities despite the highdegree of amino acid sequence homology. Assays known those skilled inthe art to measure immunomodulatory, antiproliferative, antiviral, andantimicrobial activities of one or more IFN α species may be used toevaluate hIFN polypeptides of the invention.

In vitro assays that measure secretion of prostaglandins, e.g., PGE2,may be performed to evaluate hIFN polypeptides of the invention.Prostaglandins modulate a number of CNS functions such as the generationof fever and the perception of pain, and one of the side effects ofcurrent IFN therapies is fever.

Transcriptional activity of monoPEGylated interferon-α-2a isomers isdescribed in Foser et al. Pharmacogenomics Journal 2003; 3:312-319,which is incorporated by reference herein in its entirety, usingoligonucleotide array transcript analysis. Similar assays may beperformed with hIFN polypeptides of the invention. Cell lines that maybe used in gene expression studies include but are not limited to,melanoma cell lines such as ME15. ME15 or similar cell lines may also beused to investigate functional properties of hIFN polypeptides of theinvention. Alternate assays to DNA microarrays may be performed withhIFN polypeptides of the invention to provide mRNA profiling data,differential gene expression information, or altered gene expressiondata (change in the level of a transcription or translation products).Cellular arrays may also be performed. DNA microarray or DNA chipstudies involve assembling PCR products of a group of genes or all geneswithin a genome on a solid surface in a high density format or array.General methods for array construction and use are available (see SchenaM, Shalon D, Davis R W, Brown P O., Quantitative monitoring of geneexpression patterns with a complementary DNA microarray. Science. Oct.20, 1995; 270(5235): 467-70). A DNA microarray allows the analysis ofgene expression patterns or profile of many genes to be performedsimultaneously by hybridizing the DNA microarray comprising these genesor PCR products of these genes with cDNA probes prepared from the sampleto be analyzed. DNA microarray or “chip” technology permits examinationof gene expression on a genomic scale, allowing transcription levels ofmany genes to be measured simultaneously. Methods and materials forvarious arrays and similar analyses are known to those of ordinary skillin the art.

The above compilation of references for assay methodologies is notexhaustive, and those skilled in the art will recognize other assaysuseful for testing for the desired end result. Alterations to suchassays are known to those of ordinary skill in the art.

XIII. Measurement of Potency, Functional In Vivo Half-Life, Toxicity,and Pharmacokinetic Parameters

An important aspect of the invention is the prolonged biologicalhalf-life that is obtained by construction of the hIFN polypeptide withor without conjugation of the polypeptide to a water soluble polymermoiety. The rapid decrease of hIFN polypeptide serum concentrations hasmade it important to evaluate biological responses to treatment withconjugated and non-conjugated hIFN polypeptide and variants thereof. Theconjugated and non-conjugated hIFN polypeptide and variants thereof ofthe present invention may have prolonged serum half-lives also aftersubcutaneous or i.v. administration, making it possible to measure by,e.g. ELISA method or by a primary screening assay. ELISA or RIA kitsfrom either BioSource International (Camarillo, Calif.) or DiagnosticSystems Laboratories (Webster, Tex.) may be used. Another example of anassay for the measurement of in vivo half-life of IFN or variantsthereof is described in Kozlowski et al., BioDrugs 15:419 (2001); Bailonet al., Bioconj. Chem. 12:195 (2001); Youngster et al., Current Pharm.Design 8:2139 (2002); U.S. Pat. Nos. 6,524,570; 6,250,469; 6,180,096;6,177,074; 6,042,822; 5,981,709; 5,591,974; 5,908,621; 5,738,846, whichare incorporated by reference herein. Measurement of in vivo biologicalhalf-life is carried out as described herein.

The potency and functional in vivo half-life of a hIFN polypeptidecomprising a non-naturally encoded amino acid can be determinedaccording to the protocol described in U.S. Pat. Nos. 5,711,944;5,382,657, which are incorporated by reference herein.

Pharmacokinetic parameters for a hIFN polypeptide comprising anon-naturally encoded amino acid can be evaluated in normalSprague-Dawley male rats (N=5 animals per treatment group). Animals willreceive either a single dose of 25 ug/rat iv or 50 ug/rat sc, andapproximately 5-7 blood samples will be taken according to a pre-definedtime course, generally covering about 6 hours for a hIFN polypeptidecomprising a non-naturally encoded amino acid not conjugated to a watersoluble polymer and about 4 days for a hIFN polypeptide comprising anon-naturally encoded amino acid and conjugated to a water solublepolymer. Pharmacokinetic data for hIFN polypeptides is well-studied inseveral species and can be compared directly to the data obtained forhIFN polypeptides comprising a non-naturally encoded amino acid. SeeMordenti J., et al., Pharm. Res. 8(11):1351-59 (1991) for studiesrelated to hGH.

Pharmacokinetic parameters can also be evaluated in a primate, e.g.,cynomolgus monkeys. Typically, a single injection is administered eithersubcutaneously or intravenously, and serum hIFN levels are monitoredover time.

Uno et al., which is incorporated by reference herein, in J InterferonCytokine Res. 1998 December; 18(12):1011-8 describe the induction of2′,5′ oligoadenylate synthetase in THP-1 cells in response to IFN. Inparticular, the assay described is sensitive to IFNα and may be usefulas a bioassay for serum interferon.

Animal models used to study antiviral activity of hIFN polypeptidesinclude, but are not limited to, the chimpanzee HCV (Purcell R H, FEMSMicrobiol Rev. 1994 July; 14(3):181-91), the HCV-Trimera mouse (Ilan Eet al. J Infect Dis. 2002 Jan. 15; 185(2):153-61), and the Alb-uPAtransgenic mouse models (Mercer D F et al. Nat Med. 2001 August;7(8):927-33; Kneteman, N et al (2003) 10th HCV Meeting Kyoto Japan,P-187), all of which are incorporated by reference herein. Other animalmodels may be used to evaluate side effects such as fever, depression,and pain threshold.

hIFN polypeptides of the invention may be evaluated using a human bonemarrow NOD/SCID reconstitution model. Variations to this protocol areknown to one of ordinary skill in the art. With this model, thetransplantation protocol is as follows: NOD/SCID mice are housed understerile conditions in microisolator cages. Just before and for 2 monthsafter total body irradiation mice receive acidified H₂O (pH=3). TheNOD/SCID mice are sublethally irradiated 325-350 cGy using a 137Csγ-irradiator (approximately 1 cGy/min). Cells are transplanted withhuman MNC (mononuclear cells from bone marrow or cord blood) by i.v.injection on the day of irradiation. For the reconstitution analysis, at6-8 weeks post-injection mice are bled through the tail vein, orretro-orbitally, collected into heparinized vacutubes. Peripheral bloodcells are analyzed by FACS using anti-human CD45 and anti-mouse CD45 toassess reconstitution. Mice with >0.1% human cells in the peripheralblood are considered positive.

The mice are treated with PEGASYS® or hIFN polypeptide with a dosingregimen. Various dosing regimens may be used. Mice are s.c. injectedwith drug vs. buffer, weekly dosing. On week 4, the animals aresacrificed and peripheral blood, spleen, liver, and bone marrow areharvested for analysis. The samples are processed in the followingmanner: Peripheral blood is collected into heparinized vacutubes. Thespleen and liver are removed and a single cell suspension generated.Tibia and femur bone marrow is harvested. Levels of PEGASYS® or hIFNpolypeptide are measured in blood and tissue by methods including butnot limited to, ELISA. Peled et al. in Science (1999) 283:845-848 andKim et al. in Stem Cells (1999) 17:286-294 discuss the repopulatingcells of SCID mice.

Cynomolgus monkeys are used to evaluate in vivo activity and bone marrowtoxicity of hIFN polypeptides. Induction of 2′,5′-Oligoadenylatesynthetase (2′5′-OAS) is measured in monkeys and reflects activity ofhIFN polypeptides. Bone marrow toxicity is evaluated by measuringcirculating blood cells, including but not limited to neutrophils, RBCs,and platelets as well as potentially collecting and evaluating bonemarrow.

Additional animal models for evaluating hIFN polypeptides of theinvention include animal models studying depression. Capuron, L et al.in Am. J. Psychiatry 2003; 160:1342-1344 present clinical datasuggesting a correlation between IFN-α, the HPA axis, and depression.During the first twelve weeks of IFN-α therapy, the plasmaconcentrations of adrenocorticotropic hormone (ACTH), cortisol, andinterleukin-6 (IL-6) were measured in patients both immediately beforeand one, two, and three hours after administration. The patients werealso evaluated for symptoms of major depression during the course of thestudy. It was found that patients who subsequently met symptom criteriafor major depression exhibited higher ACTH and cortisol responses (butnot IL-6 responses) to the initial administration of IFN-α than patientswho did not meet the symptom criteria.

Yamano, M. et al. JPET 2000; 292:181-187, which is incorporated byreference herein, discusses Sumiferon, a lymphoblastoid preparation ofhuman IFN-α (Sumitomo Pharmaceuticals), and YM643 (IFN-alphacon-1;Amgen) and their effects on mice. Upon dosing i.v. both compoundsinduced immobility, a depression-like behavior, in mice, as measured bythe Tail Suspension Test (TST) in a dose-dependent manner. Similarresults were achieved by s.c. and i.c.v. injection, although dose levelsand general dosing regimens differed. Imipramine (a tricyclicanti-depressant and down-regulator of the HPA axis) was found tosignificantly reduce IFN-induced immobility, whereas indomethacin (acyclooxygenase inhibitor) and naloxone (an opioid receptor antagonist)did not reduce the observed IFN-induced immobility. Yamano et al. alsoshowed that the CRF antagonist CP-154,526 blocks IFN-induced immobilityand does so in a dose-dependent manner, implicating the involvement ofthe HPA axis in IFN-α induced depression.

The Tail Suspension Test is an alternative to the “behavioral despair”or swim test in which an animal is forced to swim in a particular areawithout an escape. With the Tail Suspension Test, the animal issuspended from its tail for defined length of time, e.g., six minutes,and cannot escape. During the test, various measurements are takenincluding but not limited to, the number of times each animal entersinto an escape behavior (called an event) e.g. struggling episodes, theduration of the event, and the average strength of each event. TailSuspension Test equipment is available, for example, from LafayetteInstrument (Lafayette, Ind.).

Other studies involve animal models of other side effects found withcurrent IFN therapies, such as neutropenia. Animal studies performed maybe performed in combination with Ribavirin or another compound.

The specific activity of hIFN polypeptides in accordance with thisinvention can be determined by various assays known in the art. Thebiological activity of the hIFN polypeptide muteins, or fragmentsthereof, obtained and purified in accordance with this invention can betested by methods described or referenced herein or known to those ofordinary skill in the art.

XIV. Administration and Pharmaceutical Compositions

The polypeptides or proteins of the invention (including but not limitedto, hIFN, synthetases, proteins comprising one or more unnatural aminoacid, etc.) are optionally employed for therapeutic uses, including butnot limited to, in combination with a suitable pharmaceutical carrier.Such compositions, for example, comprise a therapeutically effectiveamount of the compound, and a pharmaceutically acceptable carrier orexcipient. Such a carrier or excipient includes, but is not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol, and/orcombinations thereof. The formulation is made to suit the mode ofadministration. In general, methods of administering proteins are knownto those of ordinary skill in the art and can be applied toadministration of the polypeptides of the invention.

Therapeutic compositions comprising one or more polypeptide of theinvention are optionally tested in one or more appropriate in vitroand/or in vivo animal models of disease, to confirm efficacy, tissuemetabolism, and to estimate dosages, according to methods known to thoseof ordinary skill in the art. In particular, dosages can be initiallydetermined by activity, stability or other suitable measures ofunnatural herein to natural amino acid homologues (including but notlimited to, comparison of a hIFN polypeptide modified to include one ormore unnatural amino acids to a natural amino acid hIFN polypeptide),i.e., in a relevant assay.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells. The unnaturalamino acid polypeptides of the invention are administered in anysuitable manner, optionally with one or more pharmaceutically acceptablecarriers. Suitable methods of administering such polypeptides in thecontext of the present invention to a patient are available, and,although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective action or reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention.

hIFN polypeptides of the invention may be administered by anyconventional route suitable for proteins or peptides, including, but notlimited to parenterally, e.g. injections including, but not limited to,subcutaneously or intravenously or any other form of injections orinfusions. Polypeptide compositions can be administered by a number ofroutes including, but not limited to oral, intravenous, intraperitoneal,intramuscular, transdermal, subcutaneous, topical, sublingual, or rectalmeans. Compositions comprising non-natural amino acid polypeptides,modified or unmodified, can also be administered via liposomes. Suchadministration routes and appropriate formulations are generally knownto those of skill in the art. The hIFN polypeptide comprising anon-naturally encoded amino acid, may be used alone or in combinationwith other suitable components such as a pharmaceutical carrier.

The hIFN polypeptide comprising a non-natural amino acid, alone or incombination with other suitable components, can also be made intoaerosol formulations (i.e., they can be “nebulized”) to be administeredvia inhalation. Aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations of hIFN can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials.

Parenteral administration and intravenous administration are preferredmethods of administration. In particular, the routes of administrationalready in use for natural amino acid homologue therapeutics (includingbut not limited to, those typically used for EPO, GH, G-CSF, GM-CSF,IFNs, interleukins, antibodies, and/or any other pharmaceuticallydelivered protein), along with formulations in current use, providepreferred routes of administration and formulation for the polypeptidesof the invention.

The dose administered to a patient, in the context of the presentinvention, is sufficient to have a beneficial therapeutic response inthe patient over time, or, including but not limited to, to inhibitinfection by a pathogen, or other appropriate activity, depending on theapplication. The dose is determined by the efficacy of the particularvector, or formulation, and the activity, stability or serum half-lifeof the unnatural amino acid polypeptide employed and the condition ofthe patient, as well as the body weight or surface area of the patientto be treated. The size of the dose is also determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, formulation, or the like in aparticular patient.

In determining the effective amount of the vector or formulation to beadministered in the treatment or prophylaxis of disease (including butnot limited to, cancers, inherited diseases, diabetes, AIDS, or thelike), the physician evaluates circulating plasma levels, formulationtoxicities, progression of the disease, and/or where relevant, theproduction of anti-unnatural amino acid polypeptide antibodies.

The dose administered, for example, to a 70 kilogram patient, istypically in the range equivalent to dosages of currently-usedtherapeutic proteins, adjusted for the altered activity or serumhalf-life of the relevant composition. The vectors or pharmaceuticalformulations of this invention can supplement treatment conditions byany known conventional therapy, including antibody administration,vaccine administration, administration of cytotoxic agents, naturalamino acid polypeptides, nucleic acids, nucleotide analogues, biologicresponse modifiers, and the like.

For administration, formulations of the present invention areadministered at a rate determined by the LD-50 or ED-50 of the relevantformulation, and/or observation of any side-effects of the unnaturalamino acid polypeptides at various concentrations, including but notlimited to, as applied to the mass and overall health of the patient.Administration can be accomplished via single or divided doses.

If a patient undergoing infusion of a formulation develops fevers,chills, or muscle aches, he/she receives the appropriate dose ofaspirin, ibuprofen, acetaminophen or other pain/fever controlling drug.Patients who experience reactions to the infusion such as fever, muscleaches, and chills are premedicated 30 minutes prior to the futureinfusions with either aspirin, acetaminophen, or, including but notlimited to, diphenhydramine. Meperidine is used for more severe chillsand muscle aches that do not quickly respond to antipyretics andantihistamines. Cell infusion is slowed or discontinued depending uponthe severity of the reaction.

Human hIFN polypeptides of the invention can be administered directly toa mammalian subject. Administration is by any of the routes normallyused for introducing hIFN polypeptide to a subject. The hIFN polypeptidecompositions according to embodiments of the present invention includethose suitable for oral, rectal, topical, inhalation (including but notlimited to, via an aerosol), buccal (including but not limited to,sub-lingual), vaginal, parenteral (including but not limited to,subcutaneous, intramuscular, intradermal, intraarticular, intrapleural,intraperitoneal, inracerebral, intraarterial, or intravenous), topical(i.e., both skin and mucosal surfaces, including airway surfaces) andtransdermal administration, although the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated. Administration can be either local or systemic. Theformulations of compounds can be presented in unit-dose or multi-dosesealed containers, such as ampoules and vials. hIFN polypeptides of theinvention can be prepared in a mixture in a unit dosage injectable form(including but not limited to, solution, suspension, or emulsion) with apharmaceutically acceptable carrier. hIFN polypeptides of the inventioncan also be administered by continuous infusion (using, including butnot limited to, minipumps such as osmotic pumps), single bolus orslow-release depot formulations.

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. Solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.

Freeze-drying is a commonly employed technique for presenting proteinswhich serves to remove water from the protein preparation of interest.Freeze-drying, or lyophilization, is a process by which the material tobe dried is first frozen and then the ice or frozen solvent is removedby sublimation in a vacuum environment. An excipient may be included inpre-lyophilized formulations to enhance stability during thefreeze-drying process and/or to improve stability of the lyophilizedproduct upon storage. Pikal, M. Biopharm. 3(9)26-30 (1990) and Arakawaet al. Pharm. Res. 8(3):285-291 (1991).

The spray drying of pharmaceuticals is also known to those of ordinaryskill in the art. For example, see Broadhead, J. et al., “The SprayDrying of Pharmaceuticals,” in Drug Dev. Ind. Pharm, 18 (11 & 12),1169-1206 (1992). In addition to small molecule pharmaceuticals, avariety of biological materials have been spray dried and these include:enzymes, sera, plasma, micro-organisms and yeasts. Spray drying is auseful technique because it can convert a liquid pharmaceuticalpreparation into a fine, dustless or agglomerated powder in a one-stepprocess. The basic technique comprises the following four steps: a)atomization of the feed solution into a spray; b) spray-air contact; c)drying of the spray; and d) separation of the dried product from thedrying air. U.S. Pat. Nos. 6,235,710 and 6,001,800, which areincorporated by reference herein, describe the preparation ofrecombinant erythropoietin by spray drying.

The pharmaceutical compositions of the invention may comprise apharmaceutically acceptable carrier, excipient, or stabilizer.Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions(including optional pharmaceutically acceptable carriers, excipients, orstabilizers) of the present invention (see, e.g., Remington'sPharmaceutical Sciences, 17^(th) ed. 1985)).

Suitable carriers include, but are not limited to, buffers containingsuccinate, phosphate, borate, HEPES, citrate, histidine, imidazole,acetate, bicarbonate, and other organic acids; antioxidants includingbut not limited to, ascorbic acid; low molecular weight polypeptidesincluding but not limited to those less than about 10 residues;proteins, including but not limited to, serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers including but not limited to,polyvinylpyrrolidone; amino acids including but not limited to, glycine,glutamine, asparagine, arginine, histidine or histidine derivatives,methionine, glutamate, or lysine; monosaccharides, disaccharides, andother carbohydrates, including but not limited to, trehalose, sucrose,glucose, mannose, or dextrins; chelating agents including but notlimited to, EDTA and edentate disodium; divalent metal ions includingbut not limited to, zinc, cobalt, or copper; sugar alcohols includingbut not limited to, mannitol or sorbitol; salt-forming counter ionsincluding but not limited to, sodium and sodium chloride; and/ornonionic surfactants including but not limited to Tween™ (including butnot limited to, Tween 80 (polysorbate 80) and Tween 20 (polysorbate 20),Pluronics™ and other pluronic acids, including but not limited to, andother pluronic acids, including but not limited to, pluronic acid F68(poloxamer 188), or PEG. Suitable surfactants include for example butare not limited to polyethers based upon poly(ethyleneoxide)-poly(propylene oxide)-poly(ethylene oxide), i.e., (PEO-PPO-PEO),or poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide),i.e., (PPO-PEO-PPO), or a combination thereof. PEO-PPO-PEO andPPO-PEO-PPO are commercially available under the trade names Pluronics™,R—Pluronics™, Tetronics™ and R-Tetronics™ (BASF Wyandotte Corp.,Wyandotte, Mich.) and are further described in U.S. Pat. No. 4,820,352incorporated herein in its entirety by reference. Otherethylene/polypropylene block polymers may be suitable surfactants. Asurfactant or a combination of surfactants may be used to stabilizePEGylated hIFN against one or more stresses including but not limited tostress that results from agitation. Some of the above may be referred toas “bulking agents.” Some may also be referred to as “tonicitymodifiers.” Antimicrobial preservatives may also be applied for productstability and antimicrobial effectiveness; suitable preservativesinclude but are not limited to, benzyl alcohol, bezalkonium chloride,metacresol, methyl/propyl parabene, cresol, and phenol, or a combinationthereof.

Formulations of interferon molecules, including but not limited to,interferon alpha and PEGylated forms of interferon alpha are describedin U.S. Pat. Nos. 5,762,923; 5,766,582; and 5,935,566, which areincorporated by reference herein in their entirety. Methods formanufacture and to test stability of interferon molecules are alsodescribed. Allen et al. International Journal of Pharmaceutics (1999)187:259-272 also discusses the formulation of a hybrid interferon-αmolecule and degradation products found under various conditions such ashigh and low pH.

hIFN polypeptides of the invention, including those linked to watersoluble polymers such as PEG can also be administered by or as part ofsustained-release systems. Sustained-release compositions include,including but not limited to, semi-permeable polymer matrices in theform of shaped articles, including but not limited to, films, ormicrocapsules. Sustained-release matrices include from biocompatiblematerials such as poly(2-hydroxyethyl methacrylate) (Langer et al., J.Biomed. Mater. Res., 15: 267-277 (1981); Langer, Chem. Tech., 12: 98-105(1982), ethylene vinyl acetate (Langer et al., supra) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988), polylactides (polylacticacid) (U.S. Pat. No. 3,773,919; EP 58,481), polyglycolide (polymer ofglycolic acid), polylactide co-glycolide (copolymers of lactic acid andglycolic acid) polyanhydrides, copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22, 547-556 (1983),poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitinsulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides,nucleic acids, polyamino acids, amino acids such as phenylalanine,tyrosine, isoleucine, polynucleotides, polyvinyl propylene,polyvinylpyrrolidone and silicone. Sustained-release compositions alsoinclude a liposomally entrapped compound. Liposomes containing thecompound are prepared by methods known per se: DE 3,218,121; Eppstein etal., Proc. Natl. Acad. Sci. U.S.A., 82: 3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci. U.S.A., 77: 4030-4034 (1980); EP 52,322; EP36,676; U.S. Pat. No. 4,619,794; EP 143,949; U.S. Pat. No. 5,021,234;Japanese Pat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545;and EP 102,324. All references and patents cited are incorporated byreference herein.

Liposomally entrapped hIFN polypeptides can be prepared by methodsdescribed in, e.g., DE 3,218,121; Eppstein et al., Proc. Natl. Acad.Sci. U.S.A., 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci.U.S.A., 77: 4030-4034 (1980); EP 52,322; EP 36,676; U.S. Pat. No.4,619,794; EP 143,949; U.S. Pat. No. 5,021,234; Japanese Pat. Appln.83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Composition and size of liposomes are well known or able to be readilydetermined empirically by one of ordinary skill in the art. Someexamples of liposomes as described in, e.g., Park J W, et al., Proc.Natl. Acad. Sci. USA 92:1327-1331 (1995); Lasic D and Papahadjopoulos D(eds): MEDICAL APPLICATIONS OF LIPOSOMES (1998); Drummond D C, et al.,Liposomal drug delivery systems for cancer therapy, in Teicher B (ed):CANCER DRUG DISCOVERY AND DEVELOPMENT (2002); Park J W, et al., Clin.Cancer Res. 8:1172-1181 (2002); Nielsen U B, et al., Biochim. Biophys.Acta 1591(1-3):109-118 (2002); Mamot C, et al., Cancer Res. 63:3154-3161 (2003). All references and patents cited are incorporated byreference herein.

The dose administered to a patient in the context of the presentinvention should be sufficient to cause a beneficial response in thesubject over time. Generally, the total pharmaceutically effectiveamount of the hIFN polypeptide of the present invention administeredparenterally per dose is in the range of about 0.01 μg/kg/day to about100 μg/kg, or about 0.05 mg/kg to about 1 mg/kg, of patient body weight,although this is subject to therapeutic discretion. The frequency ofdosing is also subject to therapeutic discretion, and may be morefrequent or less frequent than the commercially available hIFNpolypeptide products approved for use in humans. Generally, a PEGylatedhIFN polypeptide of the invention can be administered by any of theroutes of administration described above.

XV. Therapeutic Uses of hIFN Polypeptides of the Invention

The hIFN polypeptides of the invention are useful for treating a widerange of disorders.

An agonist hIFN variant may act to stimulate the immune system of amammal by increasing its immune function, whether the increase is due toantibody mediation or cell mediation, and whether the immune system isendogenous to the host treated with the hIFN polypeptide or istransplanted from a donor to the host recipient given the hIFNpolypeptide (as in bone marrow transplants). “Immune disorders” includeany condition in which the immune system of an individual has a reducedantibody or cellular response to antigens than normal, including thoseindividuals with small spleens with reduced immunity due to drug (e.g.,chemotherapeutic) treatments. Examples individuals with immune disordersinclude, e.g., elderly patients, individuals undergoing chemotherapy orradiation therapy, individuals recovering from a major illness, or aboutto undergo surgery, individuals with AIDS, Patients with congenital andacquired B-cell deficiencies such as hypogammaglobulinemia, commonvaried agammaglobulinemia, and selective immunoglobulin deficiencies(e.g., IgA deficiency, patients infected with a virus such as rabieswith an incubation time shorter than the immune response of the patient;and individuals with hereditary disorders such as diGeorge syndrome.IFNα's exhibit many immunomodulatory activities, see Zoon et al., (1986)In, The Biology of the Interferon System. Cantell and Schellenkens,Eds., Martinus Nyhoff Publishers, Amsterdam).

Administration of the hIFN products of the present invention results inany of the activities demonstrated by commercially available IFNpreparations in humans. The pharmaceutical compositions containing hIFNmay be formulated at a strength effective for administration by variousmeans to a human patient experiencing disorders that may be affected byIFN agonists or antagonists, such as but not limited to,anti-proliferatives, anti-inflammatory, or antivirals are used, eitheralone or as part of a condition or disease.

The hIFN of the present invention may thus be used to interrupt ormodulate a viral replication cycle, modulate inflammation, or asanti-proliferative agents. Among the conditions treatable by the presentinvention include HCV, HBV, and other viral infections, tumor cellgrowth or viability, and multiple sclerosis. The invention also providesfor administration of a therapeutically effective amount of anotheractive agent such as an anti-cancer chemotherapeutic agent. The amountto be given may be readily determined by one skilled in the art basedupon therapy with hIFN.

Although IFNs were first discovered by virologists, their first clinicaluse (in 1979) was as therapeutic agents for myeloma (Joshua et al.,(1997) Blood Rev. 111(4):191-200). IFNα's have since been shown to beefficacious against a myriad of diseases of viral, malignant,angiogenic, allergic, inflammatory, and fibrotic origin (Tilg, (1997)Gastroenterology. 112(3):1017-1021). It has also proven efficacious inthe treatment of metastatic renal carcinoma and chronic myeloid leukemia(Williams and Linch, (1997) Br. J. Hosp. Med. 57(9):436-439). Clinicaluses of IFNs are reviewed in Gresser (1997) J. Leukoc. Biol.61(5):567-574 and Pfeffer (1997) Semin. Oncol. 24(3 Suppl.9):S9-S63S969, which are incorporated by reference herein.

Average quantities of the hIFN may vary and in particular should bebased upon the recommendations and prescription of a qualifiedphysician. The exact amount of hIFN is a matter of preference subject tosuch factors as the exact type of condition being treated, the conditionof the patient being treated, as well as the other ingredients in thecomposition. The invention also provides for administration of atherapeutically effective amount of another active agent. The amount tobe given may be readily determined by one of ordinary skill in the artbased upon therapy with hIFN.

Pharmaceutical compositions of the invention may be manufactured in aconventional manner.

EXAMPLES

The following examples are offered to illustrate, but do not to limitthe claimed invention.

Example 1

This example describes one of the many potential sets of criteria forthe selection of preferred sites of incorporation of non-naturallyencoded amino acids into hIFN.

This example demonstrates how preferred sites within the hIFNpolypeptide were selected for introduction of a non-naturally encodedamino acid. The crystal structure with PDB ID 1RH2 and the NMR structure1ITF (twenty-four different NMR structures) were used to determinepreferred positions into which one or more non-naturally encoded aminoacids could be introduced. The coordinates for these structures areavailable from the Protein Data Bank (PDB) or via The ResearchCollaboratory for Structural Bioinformatics PDB available on the WorldWide Web at rcsb.org.

Sequence numbering used in this example is according to the amino acidsequence of mature hIFN shown in SEQ ID NO: 2. The following criteriawere used to evaluate each position of hIFN for the introduction of anon-naturally encoded amino acid: the residue (a) should not interferewith binding of hIFN based on structural analysis of crystallographicstructures of hIFN conjugated with hIFN receptor, b) should not beaffected by alanine scanning mutagenesis, (c) should be surface exposedand exhibit minimal van der Waals or hydrogen bonding interactions withsurrounding residues, (d) should be either deleted or variable in hIFNvariants, (e) would result in conservative changes upon substitutionwith a non-naturally encoded amino acid and (f) could be found in eitherhighly flexible regions (including but not limited to CD loop) orstructurally rigid regions (including but not limited to Helix B).Publications used in site evaluation include: Bioconj. Chemistry 2001(12) 195-202; Current Pharmaceutical Design 2002 (8) 2139-2157;Neuroreport 2001 (12), 857-859; BBRC 1994 (202) 1445-1451; CancerBiotherapy+Radiopharmaceuticals 1998 (vol13) 143-153; Structure 1996(14) 1453-1463; JMB 1997 (274) 661-675. In addition, furthercalculations were performed on the hIFN molecule, utilizing the Cxprogram (Pintar et al. Bioinformatics, 18, pp 980) to evaluate theextent of protrusion for each protein atom. In some embodiments, one ormore non-naturally encoded amino acid are substituted at, but notlimited to, one or more of the following positions of hIFN (as in SEQ IDNO: 2, or the corresponding amino acid in SEQ ID NO: 1, 3, or any otherIFN polypeptide): before position 1 (i.e., at the N-terminus), 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, or166 (i.e. at the carboxyl terminus). In some embodiments, one or morenon-naturally encoded amino acid are substituted at, but not limited to,one or more of the following positions of hIFN (as in SEQ ID NO: 2, orthe corresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide): before position 1 (i.e., at the N-terminus), 1, 2, 3, 4,5, 6, 7, 8, 9, 12, 13, 16, 19, 20, 22, 23, 24, 25, 26, 27, 28, 30, 31,32, 33, 34, 35, 40, 41, 42, 45, 46, 48, 49, 50, 51, 58, 61, 64, 65, 68,69, 70, 71, 73, 74, 77, 78, 79, 80, 81, 82, 83, 85, 86, 89, 90, 93, 94,96, 97, 100, 101, 103, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,117, 118, 120, 121, 124, 125, 127, 128, 129, 131, 132, 133, 134, 135,136, 137, 148, 149, 152, 153, 156, 158, 159, 160, 161, 162, 163, 164,165, or 166 (i.e. at the carboxyl terminus). In some embodiments, one ormore non-naturally encoded amino acids are incorporated in one or moreof the following positions in IFN: 6, 9, 12, 13, 16, 41, 45, 46, 48, 49,61, 64, 65, 96, 100, 101, 103, 106, 107, 108, 110, 111, 113, 114, 117,120, 121, 149, 156, 159, 160, 161 and 162 (SEQ ID NO: 2, or thecorresponding amino acids in SEQ ID NO: 1 or 3). In some embodiments,the IFN polypeptides of the invention comprise one or more non-naturallyencoded amino acids at one or more of the following positions: 100, 106,107, 108, 111, 113, 114 (SEQ ID NO: 2, or the corresponding amino acidin or the corresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide). In some embodiments, the IFN polypeptides of the inventioncomprise one or more non-naturally encoded amino acids at one or more ofthe following positions: 41, 45, 46, 48, 49 (SEQ ID NO: 2, or thecorresponding amino acid in or the corresponding amino acid in SEQ IDNO: 1, 3, or any other IFN polypeptide). In some embodiments, the IFNpolypeptides of the invention comprise one or more non-naturally encodedamino acids at one or more of the following positions: 61, 64, 65, 101,103, 110, 117, 120, 121, 149 (SEQ ID NO: 2, or the corresponding aminoacid in or the corresponding amino acid in SEQ ID NO: 1, 3, or any otherIFN polypeptide). In some embodiments, the IFN polypeptides of theinvention comprise one or more non-naturally encoded amino acids at oneor more of the following positions: 6, 9, 12, 13, 16, 96, 156, 159, 160,161, 162 (SEQ ID NO: 2, or the corresponding amino acid in or thecorresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide). In some embodiments, the IFN polypeptides of the inventioncomprise one or more non-naturally encoded amino acids at one or more ofthe following positions: 2, 3, 4, 5, 7, 8, 16, 19, 20, 40, 42, 50, 51,58, 68, 69, 70, 71, 73, 97, 105, 109, 112, 118, 148, 149, 152, 153, 158,163, 164, 165 (SEQ ID NO: 2, or the corresponding amino acid in or thecorresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide). In some embodiments, the IFN polypeptides of the inventioncomprise one or more non-naturally encoded amino acids at one or more ofthe following positions: 34, 78, 107 (SEQ ID NO: 2, or the correspondingamino acid in SEQ ID NO: 1, 3, or any other IFN polypeptide). In someembodiments, the non-naturally encoded amino acid at one or more ofthese or other positions is linked to a water soluble polymer, includingbut not limited to positions: before position 1 (i.e., at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, or 166 (i.e. at the carboxyl terminus) (SEQ ID NO:2, or the corresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide). In some embodiments, the non-naturally encoded amino acidat one or more of these positions is linked to one or more water solublepolymers, including but not limited to positions: before position 1(i.e. the N terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 13, 16, 19, 20,22, 23, 24, 25, 26, 27, 28, 30, 31, 32, 33, 34, 35, 40, 41, 42, 45, 46,48, 49, 50, 51, 58, 61, 64, 65, 68, 69, 70, 71, 73, 74, 77, 78, 79, 80,81, 82, 83, 85, 86, 89, 90, 93, 94, 96, 97, 100, 101, 103, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 117, 118, 120, 121, 124, 125,127, 128, 129, 131, 132, 133, 134, 135, 136, 137, 148, 149, 152, 153,156, 158, 159, 160, 161, 162, 163, 164, 165, 166 (i.e. at the carboxylterminus) (SEQ ID NO: 2, or the corresponding amino acid in or thecorresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide). In some embodiments, the non-naturally encoded amino acidis linked to a water soluble polymer at one or more of the followingpositions: 6, 9, 12, 13, 16, 41, 45, 46, 48, 49, 61, 64, 65, 96, 100,101, 103, 106, 107, 108, 110, 111, 113, 114, 117, 120, 121, 149, 156,159, 160, 161 and 162 (SEQ ID NO: 2, or the corresponding amino acids inSEQ ID NO: 1 or 3). In some embodiments, the one or more non-naturallyencoded amino acids at one or more of the following positions is linkedto one or more water-soluble polymer: 100, 106, 107, 108, 111, 113, 114(SEQ ID NO: 2, or the corresponding amino acid in or the correspondingamino acid in SEQ ID NO: 1, 3, or any other IFN polypeptide). In someembodiments, the one or more non-naturally encoded amino acids at one ormore of the following positions is linked to one or more water-solublepolymer: 41, 45, 46, 48, 49 (SEQ ID NO: 2, or the corresponding aminoacid in or the corresponding amino acid in SEQ ID NO: 1, 3, or any otherIFN polypeptide). In some embodiments, the one or more non-naturallyencoded amino acids at one or more of the following positions is linkedto one or more water-soluble polymer: 61, 64, 65, 101, 103, 110, 117,120, 121, 149 (SEQ ID NO: 2, or the corresponding amino acid in or thecorresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide). In some embodiments, the one or more non-naturally encodedamino acids at one or more of the following positions is linked to oneor more water-soluble polymer: 6, 9, 12, 13, 16, 96, 156, 159, 160, 161,162 (SEQ ID NO: 2, or the corresponding amino acid in or thecorresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide). In some embodiments, the one or more non-naturally encodedamino acids at one or more of the following positions is linked to oneor more water-soluble polymer: 2, 3, 4, 5, 7, 8, 16, 19, 20, 40, 42, 50,51, 58, 68, 69, 70, 71, 73, 97, 105, 109, 112, 118, 148, 149, 152, 153,158, 163, 164, 165 (SEQ ID NO: 2, or the corresponding amino acid in orthe corresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide). In some embodiments, the one or more non-naturally encodedamino acids at one or more of the following positions is linked to oneor more water-soluble polymer: 34, 78, 107 (SEQ ID NO: 2, or thecorresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide). In some embodiments, the water soluble polymer is coupledto the IFN polypeptide to a non-naturally encoded amino acid at one ormore of the following amino acid positions: 6, 9, 12, 13, 16, 41, 45,46, 48, 49, 61, 64, 65, 96, 100, 101, 103, 106, 107, 108, 110, 111, 113,114, 117, 120, 121, 149, 156, 159, 160, 161 and 162 (SEQ ID NO: 2, orthe corresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide). In some embodiments the water soluble polymer is coupledto the IFN polypeptide at one or more of the following amino acidpositions: 6, 9, 12, 13, 16, 41, 45, 46, 48, 49, 61, 64, 65, 96, 100,101, 103, 106, 107, 108, 110, 111, 113, 114, 117, 120, 121, 149, 156,159, 160, 161 and 162 (SEQ ID NO: 2, or the corresponding amino acid inSEQ ID NO: 1, 3, or any other IFN polypeptide). In some embodiments, thenon-naturally encoded amino acid at one or more of these positions islinked to one or more water soluble polymers, positions: 34, 78, 107(SEQ ID NO: 2, or the corresponding amino acid in or the correspondingamino acid in SEQ ID NO: 1, 3, or any other IFN polypeptide). In someembodiments, the IFN polypeptides of the invention comprise one or morenon-naturally encoded amino acids at one or more of the followingpositions providing an antagonist: 2, 3, 4, 5, 7, 8, 16, 19, 20, 40, 42,50, 51, 58, 68, 69, 70, 71, 73, 97, 105, 109, 112, 118, 148, 149, 152,153, 158, 163, 164, 165 (SEQ ID NO: 2, or the corresponding amino acidin or the corresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide); a hIFN polypeptide comprising one of these substitutionsmay potentially act as a weak antagonist or weak agonist depending onthe intended site selected and desired activity. Human IFN antagonistsinclude, but are not limited to, hIFN polypeptides with one or morenon-naturally encoded amino acid substitutions at 22, 23, 24, 25, 26,27, 28, 30, 31, 32, 33, 34, 35, 74, 77, 78, 79, 80, 82, 83, 85, 86, 89,90, 93, 94, 124, 125, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137,or any combinations thereof (hIFN; SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NO: 1 or 3, or any other IFN polypeptide).

In some embodiments, one or more non-naturally encoded amino acids aresubstituted at, but not limited to, one or more of the followingpositions of hIFN (as in SEQ ID NO: 2, or the corresponding amino acidsin SEQ ID NO: 1 or 3, or any other IFN polypeptide): 31, 134, 34, 38,129, 36, 122, 37, 121, 41, 125, 124, 149, 117, 39, 118, 120, 107, 108,106, 100, 111, 113, 114, 41, 45, 46, 48, 49, 61, 64, 65, 101, 103, 102,110, 117, 120, 121, 149, 96, 6, 9, 12, 13, 16, 68, 70, 109, 159, 161,156, 160, 162, 24, 27, 78, 83, 85, 87, 89, 164. In one embodiment, anon-naturally encoded amino acid is substituted at position 38 of hIFN(as in SEQ ID NO: 2, or the corresponding amino acids in SEQ ID NO: 1 or3, or any other IFN polypeptide). In some embodiments, the non-naturallyencoded amino acid at one or more of these or other positions is linkedto a water soluble polymer, including but not limited to positions: 31,134, 34, 38, 129, 36, 122, 37, 121, 41, 125, 124, 149, 117, 39, 118,120, 107, 108, 106, 100, 111, 113, 114, 41, 45, 46, 48, 49, 61, 64, 65,101, 103, 102, 110, 117, 120, 121, 149, 96, 6, 9, 12, 13, 16, 68, 70,109, 159, 161, 156, 160, 162, 24, 27, 78, 83, 85, 87, 89, 164 (as in SEQID NO: 2, or the corresponding amino acids in SEQ ID NO: 1 or 3, or anyother IFN polypeptide).

In some embodiments, one or more non-naturally encoded amino acids aresubstituted at, but not limited to, one or more of the followingpositions of hIFN (SEQ ID NO: 2 or the corresponding amino acids in SEQID NO: 1 or 3): before position 1 (i.e., at the N-terminus), 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, or 166(i.e. at the carboxyl terminus) and one or more natural amino acidsubstitutions. In some embodiments, the one or more non-naturallyencoded amino acids are coupled to a water soluble polymer. In someembodiments, the one or more non-naturally encoded amino acids arecoupled to PEG. In one embodiment, the natural amino acid substitutionis R149Y. In some embodiments, the natural amino acid substitution isR149E. In some embodiments, the natural amino acid substitution isR149S. In one embodiment, the non-natural amino acid substitution is atposition 107 and the natural amino acid substitution is R149Y. In oneembodiment, the non-natural amino acid substitution is at position 106and the natural amino acid substitution is R149Y. In some embodiments,the one or more naturally encoded amino acid substitution is at one ormore of the following positions of hIFN (SEQ ID NO: 2 or thecorresponding amino acids in SEQ ID NO: 1 or 3), including but notlimited to: 10, 16, 13, 79, 83, 85, 86, 87, 90, 91, 93, 94, 96, 120,121, 124, 125, 128, 149. In some embodiments, the one or more naturallyencoded amino acid substitution is one or more of the followingsubstitutions (SEQ ID NO: 2 or the corresponding amino acids in SEQ IDNO: 1 or 3), including but not limited to: G10E, M16R, R13E, T79R, K83Q,K83S, Y85L, T86S, E87S, Q90R, Q91E, N93Q, D94V, E96K, R120K, K121T,Q124R, R125G, L128R, R149Y, R149E, R149S. In some embodiments, thenatural amino acid substitution is at position 1 (the N-terminus). Insome embodiments, one or more non-naturally encoded amino acids aresubstituted at one or more of the following positions of hIFN (as in SEQID NO: 2, or the corresponding amino acids in other IFN's): 107, 78, 34.In some embodiments, the non-naturally encoded amino acid at one or moreof these positions is coupled to a water soluble polymer: 107, 78, 34.

One or more amino acids found in a limitin sequence may be substitutedinto a hIFN polypeptide (hybrid limitin/hIFN polypeptides). Examplesinclude but are not limited to the natural amino acid substitutionsdescribed in the previous paragraph. Alternatively, a set of amino acidsfound in an interferon polypeptide may be replaced by a set of aminoacids found in a limitin sequence. A set of amino acids may comprisecontiguous amino acids or amino acids present in different portions ofthe molecule but are involved in a structural characteristic orbiological activity of the polypeptide. The mouse limitin molecule hasan improved CFU-GM toxicity profile compared to other IFNα proteins.Alignment of human IFNα-2a with the limitin protein sequence showed 30%amino acid identity. 50% sequence conservation was also observed. Inparticular, a prominent deletion in the limitin sequence between the Cand D helices (in the loop between C and D helices) was observed. The“HV” mutant was generated with the following substitutions in hIFNα-2a(SEQ ID NO: 2): D77-D94 is replaced with the mouse limitin sequenceHERALDQLLSSLWRELQV. The “CD” mutant was generated with the followingsubstitutions in hIFNα-2a (SEQ ID NO: 2): V105-D114 with GQSAPLP. Thishybrid molecule with the loop region from limitin substituted into thehuman IFNα-2a protein (“CD” mutant) was found to have equivalentanti-viral activity as the WHO IFN standard. In addition to the one ormore limitin amino acids, the hIFN polypeptide may comprise one or morenon-naturally encoded amino acids at any one or more positions of thehIFN polypeptide. In some embodiments, the one or more non-naturallyencoded amino acids may be linked to a water soluble polymer such as PEGor bonded directly to a water soluble polymer such as PEG. In additionto the natural amino acid substitutions in the HV or the CD mutant, oneor more additional natural amino acid substitutions may be found in thehIFN polypeptide.

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 6, 16, 34, 39, 45, 46, 64, 65, 68, 78, 85, 87, 101, 107, 108,111, 114, 118, 124, 125, 145, 146, 153, 156, 96, 149 (SEQ ID NO: 2, orthe corresponding amino acid in or the corresponding amino acid in SEQID NO: 1, 3, or any other IFN polypeptide). In some embodiments, thehIFN polypeptide comprises one or more non-naturally encoded amino acidsat one or more of the following positions linked to one or morewater-soluble polymer: 6, 16, 34, 39, 45, 46, 64, 65, 68, 78, 85, 87,101, 107, 108, 111, 114, 118, 124, 125, 145, 146, 153, 156, 96, 149 (SEQID NO: 2, or the corresponding amino acid in or the corresponding aminoacid in SEQ ID NO: 1, 3, or any other IFN polypeptide). In someembodiments, the hIFN polypeptide comprises one or more non-naturallyencoded amino acids at one or more of the following positions linked toone or more water-soluble polymer: 6, 16, 34, 39, 45, 46, 64, 65, 68,78, 85, 87, 101, 107, 108, 111, 114, 118, 124, 125, 145, 146, 153, 156,96, 149 (SEQ ID NO: 2, or the corresponding amino acid in or thecorresponding amino acid in SEQ ID NO: 1, 3, or any other IFNpolypeptide) and comprises one or more naturally encoded amino acidsubstitution. In some embodiments, the hIFN polypeptide comprises one ormore non-naturally encoded amino acids at one or more of the followingpositions linked to one or more water-soluble polymer: 6, 16, 34, 39,45, 46, 64, 65, 68, 78, 85, 87, 101, 107, 108, 111, 114, 118, 124, 125,145, 146, 153, 156, 96, 149 (SEQ ID NO: 2, or the corresponding aminoacid in or the corresponding amino acid in SEQ ID NO: 1, 3, or any otherIFN polypeptide) and comprises one or more of the following naturallyencoded amino acid substitutions G10E, M16R, R13E, T79R, K83Q, K83S,Y85L, T86S, E87S, Q90R, Q91E, N93Q, D94V, E96K, R120K, K121T, Q124R,R125G, L128R, R149Y, R149E, R149S.

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 6, 16, 37, 45, 46, 78, 87, 89, 107, 108 (SEQ ID NO: 2 or thecorresponding amino acids in SEQ ID NO: 1 or 3). In some embodiments,the hIFN polypeptide comprises one or more non-naturally encoded aminoacids at one or more of the following positions: 6, 16, 37, 45, 46, 78,87, 89, 107, 108 (SEQ ID NO: 2 or the corresponding amino acids in SEQID NO: 1 or 3) that is linked to a water soluble polymer. In someembodiments, the hIFN polypeptide comprises one or more non-naturallyencoded amino acids at one or more of the following positions: 6, 16,37, 45, 46, 78, 87, 89, 107, 108 (SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NO: 1 or 3) that is bonded to a water solublepolymer.

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 6, 16, 37, 45, 46, 78, 87, 89, 107, 108 and one or more ofthe following naturally encoded amino acid substitutions: T79R, L80A,K83S, Y85L, Y85S, T86S, E87S, Q91E (SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NO: 1 or 3). In some embodiments, the hIFNpolypeptide comprises one or more non-naturally encoded amino acids atone or more of the following positions: 6, 16, 37, 45, 46, 78, 87, 89,107, 108 that is linked to a water soluble polymer and comprises one ormore of the following naturally encoded amino acid substitutions: T79R,L80A, K83S, Y85L, Y85S, T86S, E87S, Q91E (SEQ ID NO: 2 or thecorresponding amino acids in SEQ ID NO: 1 or 3). In some embodiments,the hIFN polypeptide comprises one or more non-naturally encoded aminoacids at one or more of the following positions: 6, 16, 37, 45, 46, 78,87, 89, 107, 108 that is bonded to a water soluble polymer and comprisesone or more of the following naturally encoded amino acid substitutions:T79R, L80A, K83S, Y85L, Y85S, T86S, E87S, Q91E (SEQ ID NO: 2 or thecorresponding amino acids in SEQ ID NO: 1 or 3).

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 6, 16, 37, 45, 46, 78, 87, 107, and 108 (SEQ ID NO: 2 or thecorresponding amino acids in SEQ ID NO: 1 or 3). In some embodiments,the hIFN polypeptide comprises one or more non-naturally encoded aminoacids at one or more of the following positions: 6, 16, 37, 45, 46, 78,87, 107, and 108 (SEQ ID NO: 2 or the corresponding amino acids in SEQID NO: 1 or 3) that is linked to a water soluble polymer. In someembodiments, the hIFN polypeptide comprises one or more non-naturallyencoded amino acids at one or more of the following positions: 6, 16,37, 45, 46, 78, 87, 107, and 108 (SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NO: 1 or 3) that is bonded to a water solublepolymer.

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 6, 16, 37, 45, 46, 78, 87, 107, and 108 and comprises one ormore of the following naturally encoded amino acid substitutions: T79R,K83S, Y85L, T86S, E87S, Q91E (SEQ ID NO: 2 or the corresponding aminoacids in SEQ ID NO: 1 or 3). In some embodiments, the hIFN polypeptidecomprises one or more non-naturally encoded amino acids at one or moreof the following positions: 6, 16, 37, 45, 46, 78, 87, 107, and 108 thatis linked to a water soluble polymer and comprises one or more of thefollowing naturally encoded amino acid substitutions: T79R, K83S, Y85L,T86S, E87S, Q91E (SEQ ID NO: 2 or the corresponding amino acids in SEQID NO: 1 or 3). In some embodiments, the hIFN polypeptide comprises oneor more non-naturally encoded amino acids at one or more of thefollowing positions: 6, 16, 37, 45, 46, 78, 87, 107, and 108 that isbonded to a water soluble polymer and comprises one or more of thefollowing naturally encoded amino acid substitutions: T79R, K83S, Y85L,T86S, E87S, Q91E (SEQ ID NO: 2 or the corresponding amino acids in SEQID NO: 1 or 3).

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 37, 45, 46, 89, and 107 (SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NO: 1 or 3). In some embodiments, the hIFNpolypeptide comprises one or more non-naturally encoded amino acids atone or more of the following positions: 37, 45, 46, 89, and 107 (SEQ IDNO: 2 or the corresponding amino acids in SEQ ID NO: 1 or 3) that islinked to a water soluble polymer. In some embodiments, the hIFNpolypeptide comprises one or more non-naturally encoded amino acids atone or more of the following positions: 37, 45, 46, 89, and 107 (SEQ IDNO: 2 or the corresponding amino acids in SEQ ID NO: 1 or 3) that isbonded to a water soluble polymer.

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 37, 45, 46, 89, and 107 and comprises one or more of thefollowing naturally encoded amino acid substitutions: T79R, L80A, Y85L,Y85S, E87S (SEQ ID NO: 2 or the corresponding amino acids in SEQ ID NO:1 or 3). In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 37, 45, 46, 89, and 107 that is linked to a water solublepolymer and comprises one or more of the following naturally encodedamino acid substitutions: T79R, L80A, Y85L, Y85S, E87S (SEQ ID NO: 2 orthe corresponding amino acids in SEQ ID NO: 1 or 3). In someembodiments, the hIFN polypeptide comprises one or more non-naturallyencoded amino acids at one or more of the following positions: 37, 45,46, 89, and 107 that is bonded to a water soluble polymer and comprisesone or more of the following naturally encoded amino acid substitutions:T79R, L80A, Y85L, Y85S, E87S (SEQ ID NO: 2 or the corresponding aminoacids in SEQ ID NO: 1 or 3).

In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 23, 24, 25, 26, 27, 28, 30, 31, 32, 33, 128, 129, 131, 132,133, 134, 135, 136, 137, 158, 159, 160, 161, 162, 163, 164, 165 (SEQ IDNO: 2 or the corresponding amino acids in SEQ ID NO: 1 or 3). In someembodiments, the hIFN polypeptide comprises one or more non-naturallyencoded amino acids at one or more of the following positions that islinked or bonded to a water soluble polymer: 23, 24, 25, 26, 27, 28, 30,31, 32, 33, 128, 129, 131, 132, 133, 134, 135, 136, 137, 158, 159, 160,161, 162, 163, 164, 165 (SEQ ID NO: 2 or the corresponding amino acidsin SEQ ID NO: 1 or 3). In some embodiments, the hIFN polypeptidecomprises one or more non-naturally encoded amino acids at one or moreof the following positions: 23, 24, 27, 31, 128, 131, 134, 158 (SEQ IDNO: 2 or the corresponding amino acids in SEQ ID NO: 1 or 3). In someembodiments, the hIFN polypeptide comprises one or more non-naturallyencoded amino acids at one or more of the following positions that islinked or bonded to a water soluble polymer: 23, 24, 27, 31, 128, 131,134, 158 (SEQ ID NO: 2 or the corresponding amino acids in SEQ ID NO: 1or 3). In some embodiments, the hIFN polypeptide comprises one or morenon-naturally encoded amino acids at one or more of the followingpositions: 24, 27, 31, 128, 131, 134 (SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NO: 1 or 3). In some embodiments, the hIFNpolypeptide comprises one or more non-naturally encoded amino acids atone or more of the following positions that is linked or bonded to awater soluble polymer: 24, 27, 31, 128, 131, 134 (SEQ ID NO: 2 or thecorresponding amino acids in SEQ ID NO: 1 or 3).

Example 2

This example details cloning and expression of a modified hIFNpolypeptide in E. coli.

This example demonstrates how a hIFN polypeptide including anon-naturally encoded amino acid can be expressed in E. coli. See Nagataet. al., Nature, vol. 284, 316-320 (1980) and U.S. Pat. No. 4,364,863,which is incorporated by reference. cDNA encoding the full length hIFNand the mature form of hIFN lacking the N-terminal signal sequence areshown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively. The full lengthand mature hIFN encoding cDNA is inserted into the pBAD HISc, pET20b,and pET19b expression vectors following optimization of the sequence forcloning and expression without altering amino acid sequence.

An introduced translation system that comprises an orthogonal tRNA(O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS) is used toexpress hIFN containing a non-naturally encoded amino acid. The O-RSpreferentially aminoacylates the O-tRNA with a non-naturally encodedamino acid. In turn the translation system inserts the non-naturallyencoded amino acid into hIFN, in response to an encoded selector codon.TABLE 2 O-RS and O-tRNA sequences. SEQ ID NO: 4 M. jannaschiimtRNA_(CUA) ^(Tyr) tRNA SEQ ID NO: 5 HLAD03; an optimized ambersupressor tRNA tRNA SEQ ID NO: 6 HL325A; an optimized AGGA frameshiftsupressor tRNA tRNA SEQ ID NO: 7 Aminoacyl tRNA synthetase for theincorporation of p-azido-L-phenylalanine RS p-Az-PheRS(6) SEQ ID NO: 8Aminoacyl tRNA synthetase for the incorporation ofp-benzoyl-L-phenylalanine RS p-BpaRS(1) SEQ ID NO: 9 Aminoacyl tRNAsynthetase for the incorporation of propargyl-phenylalanine RSPropargyl-PheRS SEQ ID NO: 10 Aminoacyl tRNA synthetase for theincorporation of propargyl-phenylalanine RS Propargyl-PheRS SEQ ID NO:11 Aminoacyl tRNA synthetase for the incorporation ofpropargyl-phenylalanine RS Propargyl-PheRS SEQ ID NO: 12 Aminoacyl tRNAsynthetase for the incorporation of p-azido-phenylalanine RSp-Az-PheRS(1) SEQ ID NO: 13 Aminoacyl tRNA synthetase for theincorporation of p-azido-phenylalanine RS p-Az-PheRS(3) SEQ ID NO: 14Aminoacyl tRNA synthetase for the incorporation of p-azido-phenylalanineRS p-Az-PheRS(4) SEQ ID NO: 15 Aminoacyl tRNA synthetase for theincorporation of p-azido-phenylalanine RS p-Az-PheRS(2) SEQ ID NO: 16Aminoacyl tRNA synthetase for the incorporation ofp-acetyl-phenylalanine (LW1) RS SEQ ID NO: 17 Aminoacyl tRNA synthetasefor the incorporation of p-acetyl-phenylalanine (LW5) RS SEQ ID NO: 18Aminoacyl tRNA synthetase for the incorporation ofp-acetyl-phenylalanine (LW6) RS SEQ ID NO: 19 Aminoacyl tRNA synthetasefor the incorporation of p-azido-phenylalanine (AzPheRS-5) RS SEQ ID NO:20 Aminoacyl tRNA synthetase for the incorporation ofp-azido-phenylalanine (AzPheRS-6) RS

The transformation of E. coli with plasmids containing the modified hIFNgene and the orthogonal aminoacyl tRNA synthetase/tRNA pair (specificfor the desired non-naturally encoded amino acid) allows thesite-specific incorporation of non-naturally encoded amino acid into thehIFN polypeptide. The transformed E. coli, grown at 37° C. in mediacontaining between 0.01-100 mM of the particular non-naturally encodedamino acid, expresses modified hIFN with high fidelity and efficiency.The His-tagged hIFN containing a non-naturally encoded amino acid isproduced by the E. coli host cells as inclusion bodies or aggregates.The aggregates are solubilized and affinity purified under denaturingconditions in 6M guanidine HCl. Refolding is performed by dialysis at 4°C. overnight in 50 mM TRIS-HCl, pH 8.0, 40 μM CuSO₄, and 2% (w/v)Sarkosyl. The material is then dialyzed against 20 mM TRIS-HCl, pH 8.0,100 mM NaCl, 2 mM CaCl₂, followed by removal of the His-tag. See Boisselet al., (1993) 268:15983-93. Methods for purification of hIFN are wellknown in the art and are confirmed by SDS-PAGE, Western Blot analyses,or electrospray-ionization ion trap mass spectrometry and the like.

To generate hIFN polypeptides with a 6 His tag at the N terminus, hIFNnucleotide sequences were cloned downstream of the tag. Thetransformation of E. Coli (BL21 (DE3)) with constructs containing thehIFN polynucleotide sequence and the orthogonal aminoacyl tRNAsynthetase/tRNA pair (specific for the desired non-naturally encodedamino acid) allowed site-specific incorporation of non-naturally encodedamino acid (p-acetyl-phenylalanine) into the hIFN polypeptide.

Example 3

This example details introduction of a carbonyl-containing amino acidand subsequent reaction with an aminooxy-containing PEG.

This Example demonstrates a method for the generation of a hIFNpolypeptide that incorporates a ketone-containing non-naturally encodedamino acid that is subsequently reacted with an aminooxy-containing PEGof approximately 5,000 MW. Each of the residues identified according tothe criteria of Example 1, including but not limited to, 100, 106, 107,108, 111, 113, 114 (hIFN) is separately substituted with a non-naturallyencoded amino acid having the following structure:

The sequences utilized for site-specific incorporation ofp-acetyl-phenylalanine into hIFN are SEQ ID NO: 2 (hIFN), and SEQ ID NO:4 (muttRNA), and 16, 17 or 18 (TyrRS LW1, 5, or 6) described in Example2 above.

Once modified, the hIFN polypeptide variant comprising thecarbonyl-containing amino acid is reacted with an aminooxy-containingPEG derivative of the form:R—PEG(N)—O—(CH₂)_(n)—O—NH₂where R is methyl, n is 3 and N is approximately 5,000 MW. The purifiedhIFN containing p-acetylphenylalanine dissolved at 10 mg/mL in 25 mM MES(Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mM Hepes (Sigma Chemical,St. Louis, Mo.) pH 7.0, or in 10 mM Sodium Acetate (Sigma Chemical, St.Louis, Mo.) pH 4.5, is reacted with a 10 to 100-fold excess ofaminooxy-containing PEG, and then stirred for 10-16 hours at roomtemperature (Jencks, W. J. Am. Chem. Soc. 1959, 81, pp 475). ThePEG-hIFN is then diluted into appropriate buffer for immediatepurification and analysis.

Example 4

Conjugation with a PEG consisting of a hydroxylamine group linked to thePEG via an amide linkage.

A PEG reagent having the following structure is coupled to aketone-containing non-naturally encoded amino acid using the proceduredescribed in Example 3:R—PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—O—NH₂where R=methyl, n=4 and N is approximately 20,000 MW. The reaction,purification, and analysis conditions are as described in Example 3.

Example 5

This example details the introduction of two distinct non-naturallyencoded amino acids into hIFN polypeptides.

This example demonstrates a method for the generation of a hIFNpolypeptide that incorporates non-naturally encoded amino acidcomprising a ketone functionality at two positions among the residuesidentified according to Example 1, wherein X* represents a non-naturallyencoded amino acid. The hIFN polypeptide is prepared as described inExamples 1 and 2, except that the selector codon is introduced at twodistinct sites within the nucleic acid.

Example 6

This example details conjugation of hIFN polypeptide to ahydrazide-containing PEG and subsequent in situ reduction.

A hIFN polypeptide incorporating a carbonyl-containing amino acid isprepared according to the procedure described in Examples 2 and 3. Oncemodified, a hydrazide-containing PEG having the following structure isconjugated to the hIFN polypeptide:R—PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—X—NH—NH₂where R=methyl, n=2 and N=10,000 MW and X is a carbonyl (C═O) group. Thepurified hIFN containing p-acetylphenylalanine is dissolved at between0.1-10 mg/mL in 25 mM MES (Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mMHepes (Sigma Chemical, St. Louis, Mo.) pH 7.0, or in 10 mM SodiumAcetate (Sigma Chemical, St. Louis, Mo.) pH 4.5, is reacted with a 1 to100-fold excess of hydrazide-containing PEG, and the correspondinghydrazone is reduced in situ by addition of stock 1M NaCNBH₃ (SigmaChemical, St. Louis, Mo.), dissolved in H₂O, to a final concentration of10-50 mM. Reactions are carried out in the dark at 4° C. to RT for 18-24hours. Reactions are stopped by addition of 1 M Tris (Sigma Chemical,St. Louis, Mo.) at about pH 7.6 to a final Tris concentration of 50 mMor diluted into appropriate buffer for immediate purification.

Example 7

This example details introduction of an alkyne-containing amino acidinto a hIFN polypeptide and derivatization with mPEG-azide.

Any of the residues of hIFN identified according to Example 1, includingbut not limited to, 100, 106, 107, 108, 111, 113, 114 are substitutedwith this non-naturally encoded amino acid:

The sequences utilized for site-specific incorporation ofp-propargyl-tyrosine into hIFN are SEQ ID NO: 2 (hIFN), SEQ ID NO: 4(muttRNA, M. jannaschii mtRNA_(CUA) ^(Tyr)), and 9, 10 or 11 describedin Example 2 above. The hIFN polypeptide containing the propargyltyrosine is expressed in E. coli and purified using the conditionsdescribed in Example 3.

The purified hIFN containing propargyl-tyrosine dissolved at between0.1-10 mg/mL in PB buffer (100 mM sodium phosphate, 0.15 M NaCl, pH=8)and a 10 to 1000-fold excess of an azide-containing PEG is added to thereaction mixture. A catalytic amount of CuSO₄ and Cu wire are then addedto the reaction mixture. After the mixture is incubated (including butnot limited to, about 4 hours at room temperature or 37° C., orovernight at 4° C.), H₂O is added and the mixture is filtered through adialysis membrane. The sample can be analyzed for the addition.

In this Example, the PEG has the following structure:R—PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—N₃where R is methyl, n is 4 and N is 10,000 MW.

Example 8

This example details substitution of a large, hydrophobic amino acid ina hIFN polypeptide with propargyl tyrosine.

A Phe, Trp or Tyr residue present within one the following regions ofhIFN: 1-9 (N-terminus), 10-21 (A helix), 22-39 (region between A helixand B helix), 40-75 (B helix), 76-77 (region between B helix and Chelix), 78-100 (C helix), 101-110 (region between C helix and D helix),111-132 (D helix), 133-136 (region between D and E helix), 137-155 (Ehelix), 156-165 (C-terminus), (as in SEQ ID NO: 2 or the correspondingamino acids of other IFN polypeptides), is substituted with thefollowing non-naturally encoded amino acid as described in Example 7:

Once modified, a PEG is attached to the hIFN polypeptide variantcomprising the alkyne-containing amino acid. The PEG will have thefollowing structure:Me-PEG(N)—O—(CH₂)₂—N₃and coupling procedures would follow those in Example 7. This generatesa hIFN polypeptide variant comprising a non-naturally encoded amino acidthat is approximately isosteric with one of the naturally-occurring,large hydrophobic amino acids and which is modified with a PEGderivative at a distinct site within the polypeptide.

Example 9

This example details generation of a hIFN polypeptide homodimer,heterodimer, homomultimer, or heteromultimer separated by one or morePEG tinkers.

The alkyne-containing hIFN polypeptide variant produced in Example 7 isreacted with a bifunctional PEG derivative of the form:N₃—(CH₂)_(n)—C(O)—NH—(CH₂)₂—O-PEG(N)—O—(CH₂)₂—NH—C(O)—(CH₂)_(n)—N₃where n is 4 and the PEG has an average MW of approximately 5,000, togenerate the corresponding hIFN polypeptide homodimer where the two hIFNmolecules are physically separated by PEG. In an analogous manner a hIFNpolypeptide may be coupled to one or more other polypeptides to formheterodimers, homomultimers, or heteromultimers. Coupling, purification,and analyses will be performed as in Examples 7 and 3.

Example 10

This example details coupling of a saccharide moiety to a hIFNpolypeptide.

One residue of the following is substituted with the non-natural encodedamino acid below: 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 13, 16, 19, 20, 22, 23,24, 25, 26, 27, 28, 30, 31, 32, 33, 34, 35, 40, 41, 42, 45, 46, 48, 49,50, 51, 58, 61, 64, 65, 68, 69, 70, 71, 73, 74, 77, 78, 79, 80, 81, 82,83, 85, 86, 89, 90, 93, 94, 96, 97, 100, 101, 103, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 117, 118, 120, 121, 124, 125, 127, 128,129, 131, 132, 133, 134, 135, 136, 137, 148, 149, 152, 153, 156, 158,159, 160, 161, 162, 163, 164, 165 (as in SEQ ID NO: 2, or thecorresponding amino acids of other IFN polypeptides).

Once modified, the hIFN polypeptide variant comprising thecarbonyl-containing amino acid is reacted with a β-linked aminooxyanalogue of N-acetylglucosamine (GlcNAc). The hIFN polypeptide variant(10 mg/mL) and the aminooxy saccharide (21 mM) are mixed in aqueous 100mM sodium acetate buffer (pH 5.5) and incubated at 37° C. for 7 to 26hours. A second saccharide is coupled to the first enzymatically byincubating the saccharide-conjugated hIFN polypeptide (5 mg/mL) withUDP-galactose (16 mM) and β-1,4-galacytosyltransferase (0.4 units/mL) in150 mM HEPES buffer (pH 7.4) for 48 hours at ambient temperature(Schanbacher et al. J. Biol. Chem. 1970, 245, 5057-5061).

Example 11

This example details generation of a PEGylated hIFN polypeptideantagonist.

One of the following residues, 2, 3, 4, 5, 7, 8, 16, 19, 20, 40, 42, 50,51, 58, 68, 69, 70, 71, 73, 97, 105, 109, 112, 118, 148, 149, 152, 153,158, 163, 164, 165, (hIFN; SEQ ID NO: 2 or the corresponding amino acidsin SEQ ID NO: 1 or 3) is substituted with the following non-naturallyencoded amino acid as described in Example 3; a hIFN polypeptidecomprising one of these substitutions may potentially act as a weakantagonist or weak agonist depending on the site selected and thedesired activity. One of the following residues, 22, 23, 24, 25, 26, 27,28, 30, 31, 32, 33, 34, 35, 74, 77, 78, 79, 80, 82, 83, 85, 86, 89, 90,93, 94, 124, 125, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137,(hIFN; SEQ ID NO: 2 or the corresponding amino acids in SEQ ID NO: 1 or3) is substituted with the following non-naturally encoded amino acid asdescribed in Example 3.

Once modified, the hIFN polypeptide variant comprising thecarbonyl-containing amino acid will be reacted with anaminooxy-containing PEG derivative of the form:R—PEG(N)—O—(CH₂)_(n)—O—NH₂where R is methyl, n is 4 and N is 20,000 MW to generate a hIFNpolypeptide antagonist comprising a non-naturally encoded amino acidthat is modified with a PEG derivative at a single site within thepolypeptide. Coupling, purification, and analyses are performed as inExample 3.

Example 12 Generation of a hIFN Polypeptide Homodimer, Heterodimer,Homomultimer, or Heteromultimer in Which the hIFN Molecules are LinkedDirectly

A hIFN polypeptide variant comprising the alkyne-containing amino acidcan be directly coupled to another hIFN polypeptide variant comprisingthe azido-containing amino acid, each of which comprise non-naturallyencoded amino acid substitutions at the sites described in, but notlimited to, Example 10. This will generate the corresponding hIFNpolypeptide homodimer where the two hIFN polypeptide variants arephysically joined. In an analogous manner a hIFN polypeptide may becoupled to one or more other polypeptides to form heterodimers,homomultimers, or heteromultimers. Coupling, purification, and analysesare performed as in Examples 3, 6, and 7.

Example 13

The polyalkylene glycol (P—OH) is reacted with the alkyl halide (A) toform the ether (B). In these compounds, n is an integer from one to nineand R′ can be a straight- or branched-chain, saturated or unsaturatedC1, to C20 alkyl or heteroalkyl group. R′ can also be a C3 to C7saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, asubstituted or unsubstituted aryl or heteroaryl group, or a substitutedor unsubstituted alkaryl (the alkyl is a C₁ to C₂₀ saturated orunsaturated alkyl) or heteroalkaryl group. Typically, PEG-OH ispolyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG)having a molecular weight of 800 to 40,000 Daltons (Da).

Example 14

mPEG-OH+Br—CH₂—C≡CH→mPEG-O—CH₂—C≡CH

mPEG-OH with a molecular weight of 20,000 Da (mPEG-OH 20 kDa; 2.0 g, 0.1mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL). Asolution of propargyl bromide, dissolved as an 80% weight solution inxylene (0.56 mL, 5 mmol, 50 equiv., Aldrich), and a catalytic amount ofK1 were then added to the solution and the resulting mixture was heatedto reflux for 2 hours. Water (1 mL) was then added and the solvent wasremoved under vacuum. To the residue was added CH₂Cl₂ (25 mL) and theorganic layer was separated, dried over anhydrous Na₂SO₄, and the volumewas reduced to approximately 2 mL. This CH₂Cl₂ solution was added todiethyl ether (150 mL) drop-wise. The resulting precipitate wascollected, washed with several portions of cold diethyl ether, and driedto afford propargyl-O-PEG.

Example 15

mPEG-OH+Br—(CH₂)₃—C≡CH→mPEG-O—(CH₂)₃—C≡CH

The mPEG-OH with a molecular weight of 20,000 Da (mPEG-OH 20 kDa; 2.0 g,0.1 mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL).Fifty equivalents of 5-bromo-1-pentyne (0.53 mL, 5 mmol, Aldrich) and acatalytic amount of KI were then added to the mixture. The resultingmixture was heated to reflux for 16 hours. Water (1 mL) was then addedand the solvent was removed under vacuum. To the residue was addedCH₂Cl₂ (25 mL) and the organic layer was separated, dried over anhydrousNa₂SO₄, and the volume was reduced to approximately 2 mL. This CH₂Cl₂solution was added to diethyl ether (150 mL) drop-wise. The resultingprecipitate was collected, washed with several portions of cold diethylether, and dried to afford the corresponding alkyne. 5-chloro-1-pentynemay be used in a similar reaction.

Example 16

m-HOCH₂C₆H₄OH+NaOH+Br—CH₂—C≡CH→m-HOCH₂C₆H₄O—CH₂—C≡CH  (1)m-HOCH₂C₆H₄O—CH₂—C≡CH+MsCl+N(Et)₃ →m-MsOCH₂C₆H₄O—CH₂—C≡CH  (2)m-MsOCH₂C₆H₄O—CH₂—C≡CH+LiBr→m-Br—CH₂C₆H₄O—CH₂—C≡CH  (3)mPEG-OH+m-Br—CH₂C₆H₄O—CH₂—C≡CH→mPEG-O—CH₂—C₆H₄₀—CH₂—C≡CH  (4)

To a solution of 3-hydroxybenzylalcohol (2.4 g, 20 mmol) in THF (50 mL)and water (2.5 mL) was first added powdered sodium hydroxide (1.5 g,37.5 mmol) and then a solution of propargyl bromide, dissolved as an 80%weight solution in xylene (3.36 mL, 30 mmol). The reaction mixture washeated at reflux for 6 hours. To the mixture was added 10% citric acid(2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over MgSO₄ andconcentrated to give the 3-propargyloxybenzyl alcohol.

Methanesulfonyl chloride (2.5 g, 15.7 mmol) and triethylamine (2.8 mL,20 mmol) were added to a solution of compound 3 (2.0 g, 11.0 mmol) inCH₂Cl₂ at 0° C. and the reaction was placed in the refrigerator for 16hours. A usual work-up afforded the mesylate as a pale yellow oil. Thisoil (2.4 g, 9.2 mmol) was dissolved in THF (20 mL) and LiBr (2.0 g, 23.0mmol) was added. The reaction mixture was heated to reflux for 1 hourand was then cooled to room temperature. To the mixture was added water(2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over anhydrousNa₂SO₄, and concentrated to give the desired bromide.

mPEG-OH 20 kDa (1.0 g, 0.05 mmol, Sunbio) was dissolved in THF (20 mL)and the solution was cooled in an ice bath. NaH (6 mg, 0.25 mmol) wasadded with vigorous stirring over a period of several minutes followedby addition of the bromide obtained from above (2.55 g, 11.4 mmol) and acatalytic amount of KI. The cooling bath was removed and the resultingmixture was heated to reflux for 12 hours. Water (1.0 mL) was added tothe mixture and the solvent was removed under vacuum. To the residue wasadded CH₂Cl₂ (25 mL) and the organic layer was separated, dried overanhydrous Na₂SO₄, and the volume was reduced to approximately 2 mL.Dropwise addition to an ether solution (150 mL) resulted in a whiteprecipitate, which was collected to yield the PEG derivative.

Example 17

mPEG-NH₂+X—C(O)—(CH₂)_(n)—C≡CR′→mPEG-NH—C(O)—(CH₂)_(n)—C≡CR′

The terminal alkyne-containing poly(ethylene glycol) polymers can alsobe obtained by coupling a poly(ethylene glycol) polymer containing aterminal functional group to a reactive molecule containing the alkynefunctionality as shown above. n is between 1 and 10. R′ can be H or asmall alkyl group from C1 to C4.

Example 18

HO₂C—(CH₂)₂—C≡CH+NHS+DCC→NHSO—C(O)—(CH₂)₂—C≡CH  (1)mPEG-NH₂+NHSO—C(O)—(CH₂)₂—C≡CH→mPEG-NH—C(O)—(CH₂)₂—C≡CH  (2)

4-pentynoic acid (2.943 g, 3.0 mmol) was dissolved in CH₂Cl₂ (25 mL).N-hydroxysuccinimide (3.80 g, 3.3 mmol) and DCC (4.66 g, 3.0 mmol) wereadded and the solution was stirred overnight at room temperature. Theresulting crude NHS ester 7 was used in the following reaction withoutfurther purification.

mPEG-NH₂ with a molecular weight of 5,000 Da (mPEG-NH₂, 1 g, Sunbio) wasdissolved in THF (50 mL) and the mixture was cooled to 4° C. NHS ester 7(400 mg, 0.4 mmol) was added portion-wise with vigorous stirring. Themixture was allowed to stir for 3 hours while warming to roomtemperature. Water (2 mL) was then added and the solvent was removedunder vacuum. To the residue was added CH₂Cl₂ (50 mL) and the organiclayer was separated, dried over anhydrous Na₂SO₄, and the volume wasreduced to approximately 2 mL. This CH₂Cl₂ solution was added to ether(150 mL) drop-wise. The resulting precipitate was collected and dried invacuo.

Example 19

This Example represents the preparation of the methane sulfonyl ester ofpoly(ethylene glycol), which can also be referred to as themethanesulfonate or mesylate of poly(ethylene glycol). The correspondingtosylate and the halides can be prepared by similar procedures.mPEG-OH+CH₃SO₂Cl+N(Et)₃ →mPEG-O—SO₂CH₃ →mPEG-N₃  (2)

The mPEG-OH (MW=3,400, 25 g, 10 mmol) in 150 mL of toluene wasazeotropically distilled for 2 hours under nitrogen and the solution wascooled to room temperature. 40 mL of dry CH₂Cl₂ and 2.1 mL of drytriethylamine (15 mmol) were added to the solution. The solution wascooled in an ice bath and 1.2 mL of distilled methanesulfonyl chloride(15 mmol) was added dropwise. The solution was stirred at roomtemperature under nitrogen overnight, and the reaction was quenched byadding 2 mL of absolute ethanol. The mixture was evaporated under vacuumto remove solvents, primarily those other than toluene, filtered,concentrated again under vacuum, and then precipitated into 100 mL ofdiethyl ether. The filtrate was washed with several portions of colddiethyl ether and dried in vacuo to afford the mesylate.

The mesylate (20 g, 8 mmol) was dissolved in 75 ml of THF and thesolution was cooled to 4° C. To the cooled solution was added sodiumazide (1.56 g, 24 mmol). The reaction was heated to reflux undernitrogen for 2 hours. The solvents were then evaporated and the residuediluted with CH₂Cl₂ (50 mL). The organic fraction was washed with NaClsolution and dried over anhydrous MgSO₄. The volume was reduced to 20 mland the product was precipitated by addition to 150 ml of cold dryether.

Example 20

N₃—C₆H₄—CO₂H→N₃—C₆H₄CH₂OH  (1)N₃—C₆H₄CH₂OH→Br→CH₂—C₆H₄—N₃  (2)mPEG-OH+Br—CH₂—C₆H₄—N₃ →mPEG-O—CH₂—C₆H₄—N₃  (3)

4-azidobenzyl alcohol can be produced using the method described in U.S.Pat. No. 5,998,595, which is incorporated by reference herein.Methanesulfonyl chloride (2.5 g, 15.7 mmol) and triethylamine (2.8 mL,20 mmol) were added to a solution of 4-azidobenzyl alcohol (1.75 g, 11.0mmol) in CH₂Cl₂ at 0° C. and the reaction was placed in the refrigeratorfor 16 hours. A usual work-up afforded the mesylate as a pale yellowoil. This oil (9.2 mmol) was dissolved in THF (20 mL) and LiBr (2.0 g,23.0 mmol) was added. The reaction mixture was heated to reflux for 1hour and was then cooled to room temperature. To the mixture was addedwater (2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over anhydrousNa₂SO₄, and concentrated to give the desired bromide.

mPEG-OH 20 kDa (2.0 g, 0.1 mmol, Sunbio) was treated with NaH (12 mg,0.5 mmol) in THF (35 mL) and the bromide (3.32 g, 15 mmol) was added tothe mixture along with a catalytic amount of KI. The resulting mixturewas heated to reflux for 12 hours. Water (1.0 mL) was added to themixture and the solvent was removed under vacuum. To the residue wasadded CH₂Cl₂ (25 mL) and the organic layer was separated, dried overanhydrous Na₂SO₄, and the volume was reduced to approximately 2 mL.Dropwise addition to an ether solution (150 mL) resulted in aprecipitate, which was collected to yield mPEG-O—CH₂—C₆H₄—N₃.

Example 21

NH₂—PEG-O—CH₂CH₂CO₂H+N₃—CH₂CH₂CO₂—NHS→N₃—CH₂CH₂—C(O)NH-PEG-O—CH₂CH₂CO₂H

NH₂—PEG-O—CH₂CH₂CO₂H (MW 3,400 Da, 2.0 g) was dissolved in a saturatedaqueous solution of NaHCO₃ (10 mL) and the solution was cooled to 0° C.3-azido-1-N-hydroxysuccinimido propionate (5 equiv.) was added withvigorous stirring. After 3 hours, 20 mL of H₂O was added and the mixturewas stirred for an additional 45 minutes at room temperature. The pH wasadjusted to 3 with 0.5 N H₂SO₄ and NaCl was added to a concentration ofapproximately 15 wt %. The reaction mixture was extracted with CH₂Cl₂(100 mL×3), dried over Na₂SO₄ and concentrated. After precipitation withcold diethyl ether, the product was collected by filtration and driedunder vacuum to yield the omega-carboxy-azide PEG derivative.

Example 22

mPEG-OMs+HC≡CLi→mPEG-O—CH₂—CH₂—C≡C—H

To a solution of lithium acetylide (4 equiv.), prepared as known in theart and cooled to −78° C. in THF, is added dropwise a solution ofmPEG-OMs dissolved in THF with vigorous stirring. After 3 hours, thereaction is permitted to warm to room temperature and quenched with theaddition of 1 mL of butanol. 20 mL of H₂O is then added and the mixturewas stirred for an additional 45 minutes at room temperature. The pH wasadjusted to 3 with 0.5 N H₂SO₄ and NaCl was added to a concentration ofapproximately 15 wt %. The reaction mixture was extracted with CH₂Cl₂(100 mL×3), dried over Na₂SO₄ and concentrated. After precipitation withcold diethyl ether, the product was collected by filtration and driedunder vacuum to yield the 1-(but-3-ynyloxy)-methoxypolyethylene glycol(mPEG).

Example 23

The azide- and acetylene-containing amino acids were incorporatedsite-selectively into proteins using the methods described in L. Wang,et al., (2001), Science 292:498-500, J. W. Chin et al., Science301:964-7 (2003)), J. W. Chin et al., (2002), Journal of the AmericanChemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz, (2002),Chem Bio Chem 11:1135-1137; J. W. Chin, et al., (2002), PNAS UnitedStates of America 99:11020-11024: and, L. Wang, & P. G. Schultz, (2002),Chem. Comm., 1-10. Once the amino acids were incorporated, thecycloaddition reaction was carried out with 0.01 mM protein in phosphatebuffer (PB), pH 8, in the presence of 2 mM PEG derivative, 1 mM CuSO₄,and ˜1 mg Cu-wire for 4 hours at 37° C.

The synthesis of p-Acetyl-D,L-phenylalanine (pAF) andm-PEG-hydroxylamine derivatives is performed as described below. Theracemic pAF is synthesized using the previously described procedure inZhang, Z., Smith, B. A. C., Wang, L., Brock, A., Cho, C. & Schultz, P.G., Biochemistry, (2003) 42, 6735-6746. To synthesize them-PEG-hydroxylamine derivative, the following procedures are completed.To a solution of (N-t-Boc-aminooxy)acetic acid (0.382 g, 2.0 mmol) and1,3-Diisopropylcarbodiimide (0.16 mL, 1.0 mmol) in dichloromethane (DCM,70 mL), which is stirred at room temperature (RT) for 1 hour,methoxy-polyethylene glycol amine (m-PEG-NH₂, 7.5 g, 0.25 mmol, Mt. 30K, from BioVectra) and Diisopropylethylamine (0.1 mL, 0.5 mmol) areadded. The reaction is stirred at RT for 48 hours, and then isconcentrated to about 100 mL. The mixture is added dropwise to coldether (800 mL). The t-Boc-protected product precipitated out and iscollected by filtering, washed by ether 3×100 mL. It is further purifiedby re-dissolving in DCM (100 mL) and precipitating in ether (800 mL)twice. The product is dried in vacuum, confirmed by NMR and Nihydrintest.

The deBoc of the protected product (7.0 g) obtained above is carried outin 50% TFA/DCM (40 mL) at 0° C. for 1 hour and then at RT for 1.5 hour.After removing most of TFA in vacuum, the TFA salt of the hydroxylaminederivative is converted to the HCl salt by adding 4N HCl in dioxane (1mL) to the residue. The precipitate is dissolved in DCM (50 mL) andre-precipitated in ether (800 mL). The final product is collected byfiltering, washed with ether 3×100 mL, dried in vacuum, stored undernitrogen. Other PEG (5K, 20K) hydroxylamine derivatives can besynthesized using the same procedure.

Example 24

hIFN polypeptides were isolated according to the following protocol.Variations to the refolding method have also been performed to isolatehIFN polypeptides of the invention, modifying the composition of thebuffer that receives the solubilized IFN. Additional modifications suchas the addition of Cu++ or other mild oxiding agents during or after theprocedure, altering denaturing agents or reducing agents, altering thepH to assess refolding efficiency at various pHs may be performed.Column-based refolding methods using HIC, dye-based, size-exclusion, orion-exchange resins may be used for refolding. Other modifications tosuch methods are known to those of ordinary skill in the art.

To generate hIFN polypeptides with a 6 His tag at the N terminus, hIFNnucleotide sequences were cloned downstream of the tag. Thetransformation of E. coli (BL21 (DE3)) with constructs containing thehIFN polynucleotide sequence and the orthogonal aminoacyl tRNAsynthetase/tRNA pair (specific for the desired non-naturally encodedamino acid) allowed site-specific incorporation of non-naturally encodedamino acid (p-acetyl-phenylalanine) into the hIFN polypeptide.

Inclusion Body Preparation and Refolding

Fresh or frozen E. coli host cell pellets were resuspended inapproximately 10 mL/g of 20 mM Tris pH 7.5, 200 mM NaCl, 1 mM EDTA. Thepellets were sonicated six times for 30 seconds with 1 minuteincubations on ice between each sonication. 1 mg/mL lysozyme and DNase Iwere added to each sonicated sample, and the samples were incubated for30 minutes at room temperature.

The samples were centrifuged at 12,000 rpm for 10 minutes at 4° C., andthe supernatants for each sample were removed for analysis. The pelletswere washed three times with 40 ml of chilled IB Wash Buffer 1 (20 mMTris, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1% Triton) by detaching them fromthe sides of the tubes and resuspending the pellets by sonication for 30seconds. In between each wash, the samples were centrifuged at 12,000rpm for 5 minutes at 4° C. The pellets were then resuspended/washedtwice with 40 ml of chilled IB Wash Buffer 2 (20 mM Tris, pH 7.5, 100 mMNaCl, 1 mM EDTA). In between each wash, the samples were centrifuged at12,000 rpm for 5 minutes at 4° C.

After the two sets of washes, the pellets were solubilized by douncingin 5-20 ml of Solubilization Buffer (50 mM Tris, pH 7.5, 8M Gdn-HCl).The minimum amount of buffer necessary to completely solubilize eachpellet was used. Samples were then centrifuged at 12,000 for 15 minutesat 4° C. to remove any insoluble particles, and the supernatants weretransferred into fresh tubes.

An aliquot was taken from each sample, and the aliquots were diluted 20×in Solubilization Buffer, and the OD280 for each sample was measured todetermine protein concentration. The Extinction coefficient at 280 nm is22,500 M−1 or 1.17 mg/ml−1.

The inclusion bodies were aliquoted into 3-5 ml aliquots and stored at−80° C. 0.15 mg/ml IFN final (5 mg/mL solution in 6-8M GndHCl, pH 8)were injected in three aliquots into a fast-stirring buffer (20-50 mMTris HCl, pH 8.2, 0.5 M L-arginine, 10% Glycerol or Sucrose) (plus200-250 mM Gnd from solubilized IFN) at 4° C. The injections wereperformed under the surface of the solution to avoid foaming. Afterinjection, slow stirring was used for approximately 16-24 hours at 4° C.After the completion of the refolding reaction, the samples wereprocessed further.

The samples were concentrated with Amicon Stirring cells (YM-10membrane) to approximately 1-1.5 mg/ml (15-30 ml). The samples were thendialyzed against 30 mM Tris, pH 7.8, 20 mM NaCl, 5% glycerol overnightat 4° C. The samples were diluted to 50 ml with Q HP Buffer A (10 mMTris, pH 7.5), and purification was performed on a 5 ml Q HP AKTA columnusing a gradient of 0-30% B (10 mM Tris, pH 7.5, 1 M NaCl) over fifteencolumn volumes. The column was washed with 2.5 M NaCl, followed by 1MNaCl/1M NaOH. SDS-PAGE analysis was performed on the column fractions,and the fractions containing monomeric refolded hIFN polypeptides werepooled. The pH of each pool was adjusted by adding 1/20 volume of 1MNaAc, pH 4.5, and each pool was dialyzed overnight against 20 mM NaAc,pH 4, 20 mM NaCl, 5% glycerol. The hIFN polypeptides were concentratedto >1 mg/ml for PEGylation.

PEGylation

Oxyamino-derivatized 30K PEG is added to hIFN polypeptide at a 1:12molar ratio (30 mg/mg IFN/ml), and the mixture is incubated at 28° C.for 16-48 hours. FIG. 19 shows the 30K PEG used in the conjugation.

Isolation of PEGylated hIFN Polypeptides

PEGylated hIFN polypeptides were isolated using the Source Q 30 column.The buffers used with this column were Buffer A (10 mM Tris, pH 8.0);Buffer B (10 mM Tris, pH 8.0; 1M NaCl); and Sanitization Buffer (1MNaOH; 1M NaCl). The fractions of PEGylated hIFN were pooled, dialyzedagainst the following storage buffer: 20 mM NaAcetate, pH 6, 0.005%Tween, 125 mM NaCl. The samples were then concentrated to >8 microM andstored at 4° C.

Buffer lines were primed and washed with the appropriate buffers, andthe column and sample line were sanitized with 30-40 mL SanitizationBuffer. The sample line was rinsed with Buffer A to remove any traces ofSanitization Buffer, and the line was primed for sample addition. Thecolumn was equilibrated with 5-10 column volumes of Buffer A, untilparameters such as UV and Conductivity were stable.

A sample of PEGylated hIFN polypeptide was injected onto the column, andthe column was washed with 4 column volumes of Buffer A. The gradientused was the following: 0-20% Buffer B for 20 column volumes; 100%Buffer B for 4 column volumes. 1-1.5 mL fractions were collected fromthe column and were analyzed by SDS-PAGE to determine fractions forpooling. PEG monomer fractions were pooled. The column was prepared forthe next sample by the sanitizing column and sample line with 30-40 mLSanitization Buffer.

Example 25

This example details the measurement of hIFN activity and affinity ofhIFN polypeptides for the hIFN receptor.

Biacore Studies (Receptor Binding Affinity)

The sequence for the IFNAR2 extracellular domain (consisting of 206amino acids ending with sequence LLPPGQ) was amplified from cloneMHS1011-61064 (OpenBiosystems, Huntsville, Ala.). This insert was clonedinto the pBT20 expression vector (Novagen) downstream of the T7promoter. Protein expression was induced with 0.4 mM IPTG in BL21(DE3)cells (Novagen).

Since the expressed protein was insoluble, the inclusion bodies werepurified from lysed cells and solubilized in 6M GndCl. A 5 ml aliquot(50 mg amount) was reduced with 10 mM DTT for 45 minutes at 37° C. Thenthe mixture was injected into 200 ml of refolding buffer which consistedof 50 mM Tris pH 8, 20 mM NaCl, 0.5 M Arginine, 10% glycerol at 4° C.and incubated overnight with gentle stirring.

The refolding reaction was then concentrated to 25 ml using an Amiconstirring cell, and dialyzed overnight against 20 mM Tris, pH 8, 20 mMNaCl, 10% glycerol. Monomeric refolded IFNAR ECD was purified on HP QSepharose using the AKTA FPLC system (Amersham). Purified IFNAR2 ECD wasimmobilized on CM5 Biacore chip using a lysine-specific couplingprocedure recommended by the manufacturer. About 200 RUs of functionalprotein were immobilized. Various concentrations of IFN variants inHBS-EP buffer (Biacore) were injected at a flow rate of 50 mcl/minuteover the flowcell containing immobilized IFNAR2, and a control flowcellcontaining immobilized bovine serum albumin. Sensograms generated werefit to the 1:1 interaction model to calculate k_(on), k_(off) and K_(d)values using BiaEvaluation software (Biacore). N-terminal 6-His hIFNpolypeptides comprising the non-naturally encoded amino acidp-acetyl-phenylalanine (pAF) were tested, as well as PEGylated hIFNpolypeptides comprising pAF after isolation via the methods described inExample 25. Controls utilized in these studies included IFNαA obtainedfrom Sigma, N-6 His tagged wild-type IFN, and PEGASYS®. Data collectedfor hIFN polypeptides is shown in Table 3. Non-PEGylated hIFNpolypeptide with a para-acetylphenylalanine substitution at position 149of SEQ ID NO: 2 showed altered binding kinetics in this assay (fasterk_(off)). TABLE 3 Binding parameters for IFNα-2a:IFNAR2 interaction, asdetermined by SPR IFNα2A k_(on), × 10⁻⁶ 1/M * s k_(off), 1/s K_(d), nMSIGMA WT IFN 1.9 0.02 11 N-6His WT IFN 3.5 0.02 6 PEGASYS ® 0.1 0.03 3006His K31pAF 1.3 0.07 52 6His K31pAF-30K PEG 0.09 0.07 830 6His H34pAF3.7 0.014 4 6His H34pAF-30K PEG 0.14 0.02 140 6His E107pAF 4.2 0.019 4.86His E107pAF-30K 0.1 0.025 240 PEG 6His F38L 2.8 0.053 18 6His F38S 1.70.07 42 6His G37pAF 2.4 0.027 12 6His G37pAF-30K PEG 0.12 0.049 410 6HisE41pAF 1.9 0.031 16 6His E41pAF-30K PEG 0.079 0.044 560 6His R125pAF 1.30.025 19 6His R125pAF-30K 0.055 0.018 330 PEG 6His F36S 0.01 0.013 13006His P39pAF 1.3 0.023 17 6His P39pAF-30K PEG 0.076 0.034 440 6His N65pAF2.4 0.015 7 6His N65pAF-30K PEG 0.17 0.026 150 6His T106pAF 2.1 0.0157.5 6His T106pAF-30K 0.15 0.022 140 PEG 6His L117pAF 1.9 0.015 8 6HisL117pAF-30K 0.15 0.021 140 PEG 6His R12pAF 1.75 0.014 7.9 6HisR12pAF-30K PEG 0.049 0.0165 340 6His Y122S 0.031 0.01 300 6His F27pAF3.4 0.026 7.9 6His F27pAF-30K PEG 0.045 0.0094 210 6His L110pAF 4.30.056 12.8 6His L110pAF-30K 0.16 0.026 160 PEG 6His E113pAF 1.0 0.02 196His E113pAF-30K 0.15 0.024 150 PEG 6His K134pAF 3.4 0.036 10 6HisK134pAF-30K 0.095 0.025 270 PEG 6His N45pAF 2.4 0.0155 6.5 6HisN45pAF-30K PEG 0.134 0.034 250 6His I100pAF 1.9 0.0177 9.5 6HisI100pAF-30K PEG 0.132 0.03 225 6His E78pAF 2.4 0.015 7 6His E78pAF-30KPEG 0.147 0.025 170 6His Y89pAF 2 0.018 9 6His Y89pAF-30K PEG 0.1640.027 167 6His I24pAF 2.2 0.011 5 6His I24pAF-30K PEG 0.094 0.016 1706His M16pAF 1.4 0.024 18 6His M16pAF-30K PEG 0.075 0.034 460 6His R13pAF1.3 0.018 14 6His R13pAF-30K PEG 0.049 0.027 550 6His L9pAF 1.25 0.01714 6His L9pAF-30K PEG 0.1 0.03 300 6His R120pAF 4.4 0.018 4 6HisR120pAF-30K 0.093 0.026 280 PEG 6His R149pAF 2.1 0.16 75 6HisR149pAF-30K 0.018 0.11 >5000 PEG 6His E159pAF 6.7 0.023 3.5 6HisE159pAF-30K 0.1 0.0165 160 PEG 6His K49pAF 3.6 0.035 10 6His K49pAF-30KPEG 0.23 0.038 170 6His Q46pAF 3.4 0.08 24 6His Q46pAF-30K PEG ND 6HisQ48pAF 4.6 0.078 17 6His Q48pAF-30K PEG 0.37 0.037 100 6His Q61pAF 0.80.018 21 6His Q61pAF-30K PEG 0.24 0.034 142 6His E96pAF 4.8 0.057 126His E96pAF-30K PEG 0.09 0.026 280 6His G102pAF 3.2 0.085 27 6HisG102pAF-30K 0.12 0.026 210 PEG 6His V103pAF 4.4 0.062 14 6HisV103pAF-30K 0.15 0.027 180 PEG 6His P109pAF 5.1 0.086 17 6HisP109pAF-30K 0.16 0.03 190 PEG 6His K164pAF 1.3 0.025 19 6His K164pAF-30K0.075 0.035 460 PEGMeasurement of Phosphorylated STAT1

To assess the biological activity of modified hIFNα2a polypeptides, anassay measuring phosphorylation of STAT1, a signal transducer andactivator of transcription family member, was performed using the humanmonocytic leukemia THP-1 cells. Activation of STAT1 has been shown to beessential for the antiviral activity of IFNs (Durbin J E et al. Cell1996 84:443-450). The human monocyte line THP-1 was purchased from ATCC(Manassas, Va.) and was routinely passaged in RPMI 1640, sodiumpyruvate, penicillin, streptomycin, 10% heat-inactivated fetal bovineserum and 50 uM 2-mercaptoethanol. The cells were maintained at 37° C.in a humidified atmosphere of 5% CO₂.

The THP-1 cells were starved overnight in assay media containing 1% heatinactivated charcoal/dextran treated fetal bovine serum beforestimulation with increasing concentrations of hIFN polypeptides for 30minutes at 37° C. All stimulated cells were fixed for permeabilizationand stained with the appropriate phospho-antibody as suggested bymanufacturer (Cell Signaling Technology, Beverly, Mass.). Sampleacquisition was performed on the FACS Array with acquired data analyzedon the Flowjo software (Tree Star. Inc., Ashland, Oreg.). EC₅₀ valueswere derived from dose response curves plotted with mean fluorescentintensity against protein concentration utilizing SigmaPlot. (FIG. 2)

Additional controls were performed to confirm that the IFN inducedphosphorylation of STAT1 was selective for the IFNα receptor 2 (IFNAR2).Serum starved THP-1 were pre-incubated with increasing concentrations ofIFNAR2 extracellular domain (ECDR2) for 15 minutes at 37° C. before theaddition of IFN at an EC₈₀ dose. The extracellular domain of the IFNαreceptor 2 was found to compete effectively against IFNαA stimulatedSTAT1 phosphorylation indicating that IFNαA induced phosphorylation ofSTAT1 is selective for the IFNAR2. FIG. 3 shows that ECDR2 competes withIFNαA induced pSTAT activity.

A potential contaminant after purification of hIFN polypeptides isendotoxin. To determine if endotoxin affects the phosphorylation ofSTAT1, a range of endotoxin levels were tested in this cellular assay.FIG. 4 shows that endotoxin did not have a substantial effect on theassay at the levels tested. The endotoxin concentration range tested (asshown) provided comparable MFI (PE) to a control sample containing noendotoxin.

Alternatively, phosphorylation of STAT1 may be measured with RayBio®Cell-Based Stat1 (Tyr701) ELISA kit (Raybiotech, Norcross, Ga.). Withthis kit, the assay is performed in 96 well plates and has acolorimetric readout measurable by a spectrophotometric plate reader.

Measurement of Anti-Proliferative Activity

hIFN polypeptides were also assayed for anti-proliferative activity. Aprominent effect of IFNα's is their ability to inhibit cell growth,which is of major importance in determining anti-tumor action. The humanlymphoblastoid Daudi cell line has proven to be extremely sensitive toIFNα's, and it has been used to measure antiproliferative activity inmany IFNα's and derived hybrid polypeptides (Meister et al., J GenVirol. (1986) August; 67 (Pt 8):1633-43). Use of this cell line has beenfacilitated by its ability to be grown in suspension cultures (Evingerand Pestka, (1981) Methods Enzymol. 79:362-368).

The human Daudi B cell line was purchased from ATCC (Manassas, Va.) andgrown in RPMI 1640 supplemented with sodium pyruvate, penicillin,streptomycin (Invitrogen, Carlsbad, San Diego) and 10% heat inactivatedfetal bovine serum (Hyclone, Logan, Utah). The cell culture wasmaintained at 37° C. in a humidified atmosphere of 5% CO₂.

The Daudi cells were plated at a density of 1×10⁴ cells/well in aflat-bottom, 96-well plate. The cells were activated with increasingconcentration of IFNα2A in triplicates per dose concentration. Followinga 4-day incubation period at 37° C. with 5% CO₂, 40 ul of CellTiter 96Aqueous One solution Reagent (Promega Corporation, Madison, Wis.) wasadded to each well, and the culture was allowed to incubate for anadditional 3 hours. Absorbance was read at 490 nm using a Spectromax.EC₅₀s were obtained from dose response curves plotted with OD490 nm(average of triplicates) against protein concentration with SigmaPlot.See FIG. 5.

Since a potential contaminant after purification of hIFN polypeptides isendotoxin, a range of endotoxin levels were tested in theanti-proliferation assay. FIG. 6 shows that endotoxin did not have asubstantial affect on the assay at the levels tested.

Measurement of Phosphorylated Tyk2

Kawamoto et al. in Experimental Hematology 2004 32:797-805 and Ishida etal. Experimental Hematology 2005 33:495-503, which are incorporated byreference herein, discuss signaling differences between limitin andIFN-α involving Tyk2, CrkL, CrkII, and Daxx and side effects of IFN.Assays measuring phosphorylation of CrkL, CrkII and Tyk2 may be used toevaluate hIFN polypeptides of the invention. Evaluating thephosphorylation of CrkII in U-266 may be performed as described byPlatanias et al. in Experimental Hematology 1999; 27:1315-1321, which isincorporated by reference herein. For measuring the phosphorylation ofTyk2, the human U266B1 cells (ATCC, Manassas, Va.) were maintained inRPMI 1640 supplemented with sodium pyruvate, penicillin, streptomycin(Invitrogen, Carlsbad, San Diego) and 15% heat inactivated fetal bovineserum. The cell culture was maintained at 37° C. in a humidifiedatmosphere of 5% CO₂. Serum starved U266 cells were activated at 37° C.with IFNα2A for 5 minutes when measuring Tyk2. All stimulated cells werefixed, permeabilized and stained with the appropriate phospho-antibodyas suggested by the manufacturer (Cell Signaling Technology, Beverly,Mass.). Sample acquisition was performed on the FACS Array with acquireddata analyzed on the Flowjo software (Tree Star Inc., Ashland, Oreg.).EC₅₀ values were derived from dose response curves plotted with meanfluorescent intensity against protein concentration utilizing SigmaPlot.Competition assays with the extracellular domain of IFNAR2 are usefulfor determining specificity. See FIG. 8 for Tyk2.

Table 4 and 5 summarize data obtained with hIFN polypeptides that areIFNα2a variants. pSTAT1 data, anti-proliferation data, K_(d) valuesobtained from affinity studies with the extracellular domain of IFNAR2are shown. hIFN polypeptides comprising the non-naturally encoded aminoacid p-acetyl-phenylalanine (pAF) were tested, as well as PEGylated hIFNpolypeptides comprising pAF. Controls utilized in these studies includedWHO IFNα2A, IFNαA obtained from Sigma, and PEGASYS®. TABLE 4 pSTAT1Proliferation Kd IFNα2A Variants EC50 (pM) IC50 (pM) (nM) WHO IFNα2A16.0 ± 5.3 (n = 3) 0.26 ± 0.05 (n = 4) ND Sigma IFNαA 14.1 ± 1.7 (n = 3)0.33 ± 0.17 (n = 5) 11 Infergen ND 0.23 ± 0.04 (n = 2) PEGASYS ® 478.7 ±183.2 (n = 12) 11.3 ± 2.2 (n = 8) 300 6His-IFNα2A 17.6 (n = 1) 0.3 ± 0.4(n = 3) 6 6His IFNα-2a + limitin 62.7 0.54 loop (pool) 6HisC1G/C98 30.00.90 IFNα-2a + limitin loop (pool) 6His IFNα-2a + limitin 10 loop(select) 6HisC1G/C98 34.8 11 IFNα-2a + limitin loop (select) 6His-F36SND 64.3 (n = 1) 1300 6His-F38L 13 (n = 1) 0.2 (n = 1) 18 6His-F38S 18 (n= 1) 0.2 (n = 1) 42 6His-L9pAF ND 0.5 (n = 1) 14 6His-L9pAF-30K 430 (n= 1) 12.00 (n = 1) 300 6His-R12pAF ND 0.6 (n = 1) 8 6His-R12pAF-30K 660(n = 1) 16.7 ± 3.0 (n = 2) 340 6His-R13pAF ND 0.5 (n = 1) 146His-R13pAF-30K 807 (n = 1) >32.55 ± 1.2 (n = 2) 550 6His-M16pAF ND 0.35(n = 1) 18 6His-M16pAF-30K 202 (n = 1) 8.3 (n = 1) 460 6His-I24pAF ND0.19 (n = 1) 5 6His-I24pAF-30K 35 (n = 1) 2.2 (n = 1) 170 6His-F27pAF ND0 3 (n = 1) 8 6His-F27pAF-30K 316 (n = 1) 5.6 (n = 1) 210 6His-K31pAF 49(n = 1) 0.6 ± 0.1 (n = 2) 52 6His-K31pAF-30K 299 ± 34 (n = 2) 4.8 ± 2.3(n = 2) 830 6His-H34pAF 120 ± 122 (n = 2) 0.2 (n = 1) 4 6His-H34pAF-30K161 ± 175 (n = 3) 0.9 ± 0.1 (n = 2) 140 6His-G37pAF 21.8 (n = 1) 0.3 (n= 1) 12 6His-G37pAF-30K 193 (n = 1) 2.2 (n = 1) 410 6His-P39pAF ND 0.3(n = 1) 17 6His-P39pAF-30K 253.3 ± 241.2 (n = 3) 12.8 ± 1.4 (n = 2) 4406His-E41pAF 19.0 (n = 1) 0.2 (n = 1) 16 6His-E41pAF-30K 120.5 ± 31.8 (n= 2) 2.6 (n = 1) 560 6His-N45pAF ND 0.5 (n = 1) 7 6His-N45pAF-30K 87 (n= 1) 7.1 ± 0.7 (n = 2) 250 6His-Q48pAF 0.24 17 6His-Q48PAF-30K 119 3.00100 6His-K49pAF 0.13 10 6His-K49pAF-30K 134 3.20 170 6His-Q61pAF 1.50 216His-Q61pAF-30K >88900 no inhibition 140 6His-N65pAF 11 7 (n = 1) 0.7 (n= 1) 7 6His-N65pAF-30K >2865 ± 2908 (n = 2) 146 (n = 1) 150 6His-E78pAFND 0.13 (n = 1) 7 6His-E78pAF-30K 45.0 (n = 1) 1.9 ± 0.1 (n = 2) 1706His-Y89pAF ND 0.13 (n = 1) 9 6His-Y89pAF-30K 244 (n = 1) 4.4 ± 0.8 (n =2) 167 6His-E96pAF 0.55 12 6His-E96pAF-30K 2190 21.1 280 6His-I100pAF ND0.4 (n = 1) 10 6His-I100pAF-30K 130 (n = 1) 2.4 ± 0.1 (n = 2) 2256His-G102pAF 1.4 27 6His-G102pAF-30K 981 5.4 210 6His-V103pAF 0.6 146His-V103pAF-30K 190 3.6 180 6His-T106pAF ND 0.08 (n = 1) 86His-T106pAF-30K 136 (n = 1) 1.7 (n = 1) 140 6His-E107pAF 30.8 (n = 1)0.2 (n = 1) 5 6His-E107pAF-30K 106.7 ± 41.5 (n = 2) 3.1 ± 0.4 (n = 2)240 6His-E107pAF-B2 0.2 (n = 1) 6His-E107pAF- 34.8 (n = 1) 3.0 (n = 1)250 30K-B2 6His-P109pAF 0.47 17 6His-P109pAF-30K 454 4.5 1906His-L110pAF ND 0 5 (n = 1) 13 6His-L110pAF-30K 133 ± 116 (n = 2) 2.7 ±0.8 (n = 2) 167.5 + 10.6 (n = 2) 6His-E113pAF ND 0.7 (n = 1) 196His-E113pAF-30K 262 ± 132 (n = 2) 3.7 (n = 1) 150 6His-L117pAF ND 0.2(n = 1) 8 6His-L117pAF-30K 242 (n = 1) 4.0 (n = 1) 140 6His-R120pAF ND4.0 (n = 1) 4 6His-R120pAF-30K >1,000 no inhibition 280 6HisY122S ND11.2 (n = 1) 300 6His-R125pAF 16.0 (n = 1) 0.5 (n = 1) 196His-R125pAF-30K 225 (n = 1) 1.7 (n = 1) 330 6His-K134pAF ND 0.25 (n= 1) 10 6His-K134pAF-30K 108 ± 47 (n = 2) 2.6 (n = 1) 270 6His-R149pAFND 0.85 (n = 1) 75 6His-R149pAF-30K >29.4 >70 (n = 1) >5000 6His-E159pAFND 0.2 (n = 1) 3.5 6His-E159pAF-30K 384 (n = 1) 16.9 (n = 1) 160ND = not done

IFNα2A Variants pSTAT1 % Proliferation % WHO IFNα2A 3.34 2.33 SigmaIFNαA 2.95 2.93 Infergen ND 1.78 PEGASYS ® 100.00 100.00 6His-IFNα2A3.68 2.50 6His IFNα-2a + limitin loop (pool) 13.1 4.79 6HisC1G/C98IFNα-2a + limitin loop 6.27 7.99 (pool) 6His IFNα-2a + limitin loop(select) 6HisC1G/C98 IFNα-2a + limitin loop 7.27 (select) 6His-F36S ND570.92 6His-F38L 2.72 1.78 6His-F38S 3.76 1.78 6His-L9pAF 4.446His-L9pAF-30K 89.83 106.50 6His-R12pAF 5.33 6His-R12pAF-30K 137.88147.84 6His-R13pAF 4.53 6His-R13pAF-30K 168.59 >289.01 6His-M16pAF 3.116His-M16pAF-30K 42.2 73.70 6His-I24pAF 1.69 6His-I24pAF-30K 7.31 19.536His-F27pAF 2.66 6His-F27pAF-30K 66.02 49.72 6His-K31pAF 10.24 5.336His-K31pAF-30K 62.47 42.62 6His-H34pAF 25.04 1.78 6His-H34pAF-30K 33.647.99 6His-G37pAF 4.55 2.66 6His-G37pAF-30K 40.32 19.53 6His-P39pAF 2.666His-P39pAF-30K 52.91 113.65 6His-E41pAF 3.97 1.78 6His-E41pAF-30K 25.1723.09 6His-N45pAF 4.44 6His-N45pAF-30K 18.24 63.04 6His-Q48pAF 2.16His-Q48pAF-30K 24.86 26.6 6His-K49pAF 1.2 6His-K49pAF-30K 27.99 28.46His-Q61pAF 13.3 6His-Q61pAF-30K >18572 no inhibition 6His-N65pAF 2.446.22 6His-N65pAF-30K >532.04 1296.34 6His-E78pAF 1.15 6His-E78pAF-30K9.40 16.87 6His-Y89pAF 1.15 6His-Y89pAF-30K 50.97 38.62 6His-E96pAF 4.876His-E96pAF-30K 457.52 186.73 6His-I100pAF 3.55 6His-I100pAF-30K 27.1621.31 6His-G102pAF 12.40 6His-G102pAF-30K 204.94 47.90 6His-V103pAF 5.306His-V103pAF-30K 39.69 32.00 6His-T106pAF 0.71 6His-T106pAF-30K 28.4115.09 6His-E107pAF 6.43 1.78 6His-E107pAF-30K 22.28 27.526His-E107pAF-B2 1.78 6His-E107pAF-30K-B2 7.27 26.64 6His-P109pAF 4.166His-P109pAF-30K 94.85 39.82 6His-L110pAF 4.44 6His-L110pAF-30K 27.8223.97 6His-E113pAF 6.22 6His-E113pAF-30K 54.74 32.85 6His-L117pAF 1.786His-L117pAF-30K 50.56 35.52 6His-R120pAF 35.5 6His-R120pAF-30K >208.91no inhibition 6HisY122S 99.45 6His-R125pAF 3.34 4.44 6His-R125pAF-30K47.01 15.09 6His-K134pAF 2.22 6His-K134pAF-30K 22.52 23.09 6His-R149pAF7.55 6His-R149pAF-30K >6.14 >621.5 6His-E159pAF 1.78 6His-E159pAF-30K80.22 150.1Colony Formation Assays

Colony formation assays such as those described by Giron-Michel, J. inLeukemia 2002 16:1135-1142 may be used to evaluate proliferation ofprogenitor cells by hIFN polypeptides of the invention. Cord blood maybe used in colony formation assays.

Evaluation of the Toxicity of hIFN Polypeptides on Human Myeloid andErythroid Progenitors

Using a methylcelluose-based in vitro colony forming assay, thehematopoietic toxicity of ten compounds (nine hIFN polypeptides andPEGASYS®) was tested.

Cells: Normal human bone marrow light density cells (Poietics Inc.,Maryland) were stored at −152° C. until required for the assay. On theday of the experiment, the cells were thawed rapidly at 37° C., thecontents of the vial were diluted in 10 mls of Iscove's mediumcontaining 2% fetal bovine serum and washed by centrifugation. Thesupernatant was discarded, and the cell pellet resuspended in a knownvolume of Iscove's medium containing 2% FBS. A cell count (3% glacialacetic acid) and viability assessment by Trypan Blue exclusion wasperformed.

Test samples: Compounds were 40× stocks in a buffer of 20 mM NaAc, 50 mMNaCl, 5% glycerol, pH 6.0. Dilutions of each compound were prepared withthe same buffer to generate stock concentrations of 144, 72, 36, 18, and9 ug/ml. When added to methylcellulose, the buffer was at a finalconcentration of 2.5% and compound concentrations ranged from 3.6-0.225ug/ml. A control containing 50 EU/ml endotoxin in a buffer of 20 mMNaAc, 50 mM NaCl, 5% glycerol, pH 6.0 was diluted with the same buffer1:40 into methylcellulose to examine the effects of similar levels ofendotoxin.

Method: Clonogenic progenitors of the erythroid (CFU-E and BFU-E),granulocyte-monocyte (CFU-GM) and multipotential (CFU-GEMM) lineageswere assessed in methylcellulose-based medium (MethoCult™ 4434)containing saturating concentrations of the cytokines SCF (Stem CellFactor; 50 ng/mL), GM-CSF (Granulocyte Macrophage Colony StimulatingFactor; 10 ng/mL), IL-3 (Interleukin 3; 10 ng/mL), and EPO(Erythropoietin; 3 U/mL). CFU-E is a small erythroid colony derived fromthe most mature erythroid colony forming cells. It contains one to twoclusters with a total number of 8-200 erythroblasts. BFU-E is a largererythroid colony derived from a more primitive cell. It contains greaterthan 200 erythroblasts. CFU-GM is a colony that is derived from a colonyforming cell capable of producing colonies with forty or moregranulocyte-monocyte and/or macrophage cells. CFU-GEMM is a colony thatcontains cells from more than one lineage. It is derived from the mostprimitive colony forming cell and contains erythroid cells as well astwenty or more granulocytes, macrophages, and megakaryocytes. Ascontrols, cultures were also set-up containing vehicle (20 mM NaAc, 50mM NaCl, 5% glycerol, pH 6.0), vehicle containing endotoxin, and variousconcentrations of 5-Fluorouracil (a known myelosuppressive compound).Standard cultures containing no buffer or compound were alsoestablished.

Clonogenic progenitors of the erythroid (CFU-E and BFU-E),granulocyte-monocyte/myeloid (CFU-GM) and multipotential (CFU-GEMM)lineages were set up in the methylcellulose-based media described. Thecompounds were added to the MethoCult™ to give final concentrations of3.6, 1.8, 0.9, 0.45, and 0.225 ug/ml. Vehicle control culturescontaining no compound but equivalent concentrations of vehicle buffer,endotoxin control cultures containing no compound but equivalentconcentrations of endotoxin, as well as standard controls containing nocompounds or vehicle buffer were also initiated. In addition, cultureswere initiated with 5-Fluorouracil (5-FU) at 5, 1, 0.5, and 0.1 ug/ml toserve as positive controls for toxicity. All cultures were set up intriplicate at 1×10⁴ cells per culture. Following 14 days in culture, thecolonies were assessed and scored. The colonies were divided into thefollowing categories, based on size and morphology: CFU-E, BFU-E,CFU-GM, and CFU-GEMM.

Results and analysis: Triplicate cultures for CFU-E, BFU-E, CFU-GM, andCFU-GEMM were enumerated. In addition, the distribution of colony typesas well as general colony and cellular morphology were analyzed. Thevariance in colony number detected in replicate cultures isrepresentative of the coefficient of variation for colony enumeration.The results are shown in Table 6. For statistical analysis, allcompounds were compared to the vehicle control. Standard t-tests wereperformed to assess if there was a difference in the number of coloniesgenerated between control and treated cultures. Due to the potentialsubjectivity of colony enumberation, a p value of less than 0.01 isdeemed significant. TABLE 6 Total CFU-E BFU-E Erythroid CFU-GM CFU-GEMMTotal CFC Standard 10 +/− 1 43 +/− 7  53 +/− 8  61 +/− 5  1 +/− 1 115+/− 7  Vehicle control 10 +/− 3 40 +/− 8  50 +/− 6  61 +/− 9  1 +/− 1112 +/− 15  Endotoxin 10 +/− 4 41 +/− 8  51 +/− 11 57 +/− 9  1 +/− 1 109+/− 20  control 5-FU   5 ug/ml ND* ND# ND** ND** ND ND**   1 ug/ml 13+/− 2 4 +/− 2* 17 +/− 2#  2 +/− 1** ND  19 +/− 3** 0.5 ug/ml 12 +/− 3 24+/− 7  36 +/− 9  20 +/− 5* ND 56 +/− 8* 0.1 ug/ml 12 +/− 3 36 +/− 6  48+/− 9  46 +/− 5  1 +/− 1 95 +/− 13 PEGASYS ® 3.6 ug/ml 10 +/− 2 5 +/− 3*15 +/− 5* 16 +/− 3* ND 31 +/− 8# 1.8 ug/ml 10 +/− 2 9 +/− 3* 19 +/− 4*19 +/− 6* ND  38 +/− 10* 0.9 ug/ml 11 +/− 4 10 +/− 2*  21 +/− 4* 21 +/−3* ND 42 +/− 1* 0.45 ug/ml  10 +/− 4 12 +/− 3*  22 +/− 2* 27 +/− 3* ND49 +/− 2* 0.225 ug/ml  13 +/− 3 19 +/− 3$  32 +/− 1$ 31 +/− 7  ND 63 +/−7$ 6His M16pAF- 30K 3.6 ug/ml 12 +/− 2 8 +/− 1* 20 +/− 1# 14 +/− 4* ND34 +/− 5# 1.8 ug/ml 12 +/− 2 7 +/− 2* 19 +/− 3* 12 +/− 3# ND 31 +/− 2#0.9 ug/ml 11 +/− 3 11 +/− 3*  22 +/− 6* 19 +/− 1* ND 41 +/− 7* 0.45ug/ml  11 +/− 1 17 +/− 10  28 +/− 10 31 +/− 4$ ND  59 +/− 13$ 0.225ug/ml  12 +/− 4 19 +/− 3  31 +/− 7  25 +/− 8$ ND  56 +/− 15$ 6HisI24pAF-30K 3.6 ug/ml 11 +/− 2 1 +/− 1# 12 +/− 3#  7 +/− 4# ND 19 +/− 6#1.8 ug/ml 10 +/− 4 2 +/− 3* 12 +/− 3#  7 +/− 2# ND  19 +/− 5** 0.9 ug/ml11 +/− 3 2 +/− 2# 13 +/− 4#  9 +/− 4# ND 22 +/− 8# 0.45 ug/ml  10 +/− 34 +/− 1* 14 +/− 3# 10 +/− 3# ND 24 +/− 4# 0.225 ug/ml  10 +/− 2 11 +/−2*  21 +/− 3* 12 +/− 5* ND 33 +/− 5# 6 His F27pAF- 30K 3.6 ug/ml 15 +/−5 3 +/− 1# 18 +/− 6*  8 +/− 2# ND 26 +/− 4# 1.8 ug/ml 13 +/− 2 3 +/− 1*16 +/− 2# 11 +/− 3# ND 27 +/− 4# 0.9 ug/ml 18 +/− 5 7 +/− 2* 25 +/− 6$12 +/− 3# ND 37 +/− 9* 0.45 ug/ml  11 +/− 4 11 +/− 4*  22 +/− 6* 16 +/−4* ND  38 +/− 10* 0.225 ug/ml  14 +/− 4 12 +/− 2*  26 +/− 6$ 15 +/− 3*ND 41 +/− 9* 6His N45pAF- 30K 3.6 ug/ml 12 +/− 1 4 +/− 1* 16 +/− 1#  9+/− 4# ND 25 +/− 3# 1.8 ug/ml 13 +/− 3 4 +/− 2* 17 +/− 4* 12 +/− 2# ND29 +/− 6# 0.9 ug/ml 16 +/− 3 8 +/− 4* 24 +/− 7$ 18 +/− 4* ND  42 +/− 10*0.45 ug/ml  12 +/− 2 11 +/− 4*  23 +/− 5* 18 +/− 3* ND 41 +/− 5* 0.225ug/ml  10 +/− 3 15 +/− 5$  25 +/− 5$ 19 +/− 4* ND 44 +/− 7* 6His N65pAF-30K 3.6 ug/ml 13 +/− 4 12 +/− 7$  25 +/− 8$ 20 +/− 5* ND  45 +/− 11* 1.8ug/ml 17 +/− 2 12 +/− 3*  29 +/− 4$ 20 +/− 5* ND 49 +/− 7* 0.9 ug/ml 13+/− 3 12 +/− 2*  25 +/− 4* 28 +/− 4$ ND 53 +/− 8* 0.45 ug/ml  14 +/− 312 +/− 1*  26 +/− 3* 29 +/− 8$ ND  55 +/− 11$ 0.225 ug/ml  12 +/− 3 22+/− 3  34 +/− 5  27 +/− 5* ND 61 +/− 8$ 6His E78pAF- 30K 3.6 ug/ml 12+/− 3 4 +/− 4* 16 +/− 6*  3 +/− 3# ND 19 +/− 8# 1.8 ug/ml 13 +/− 3 3 +/−3* 16 +/− 4* 14 +/− 4* ND 30 +/− 8# 0.9 ug/ml 11 +/− 2 7 +/− 4* 18 +/−4* 19 +/− 6* ND 37 +/− 9* 0.45 ug/ml  13 +/− 3 7 +/− 3* 20 +/− 2* 22 +/−4* ND 42 +/− 3* 0.225 ug/ml  10 +/− 1 12 +/− 2*  22 +/− 1* 22 +/− 4* ND44 +/− 3* 6His E107pAF- 30K 3.6 ug/ml  7 +/− 3 2 +/− 3*  9 +/− 6*  8 +/−1# ND  17 +/− 6** 1.8 ug/ml 12 +/− 3 3 +/− 1# 15 +/− 3# 12 +/− 2# ND 27+/− 5# 0.9 ug/ml 15 +/− 4 4 +/− 1* 18 +/− 4* 13 +/− 3* ND 31 +/− 6# 0.45ug/ml  15 +/− 2 6 +/− 4* 21 +/− 4* 18 +/− 6* ND 39 +/− 6* 0.225 ug/ml 12 +/− 4 9 +/− 1* 21 +/− 4* 18 +/− 2* ND 39 +/− 2# 6His R125pAF- 30K 3.6ug/ml 13 +/− 1 4 +/− 2* 17 +/− 2# 12 +/− 3# ND 29 +/− 4# 1.8 ug/ml 12+/− 4 4 +/− 2* 16 +/− 3# 13 +/− 3# ND 29 +/− 5# 0.9 ug/ml 10 +/− 1 5 +/−3* 15 +/− 3# 16 +/− 4* ND 31 +/− 6# 0.45 ug/ml  15 +/− 2 6 +/− 4* 21 +/−5* 21 +/− 2* ND 42 +/− 6* 0.225 ug/ml  11 +/− 2 10 +/− 5*  21 +/− 5* 17+/− 5* ND 38 +/− 9* 6His K134pAF- 30K 3.6 ug/ml  6 +/− 3 1 +/− 1#  7 +/−2** 10 +/− 2# ND  17 +/− 4** 1.8 ug/ml 10 +/− 3 2 +/− 2# 12 +/− 4# 11+/− 3# ND  23 +/− 3** 0.9 ug/ml  8 +/− 3 1 +/− 2#  9 +/− 3** 11 +/− 3#ND 20 +/− 6# 0.45 ug/ml  14 +/− 2 4 +/− 2* 18 +/− 3* 12 +/− 3# ND 30 +/−5# 0.225 ug/ml  10 +/− 4 6 +/− 2* 16 +/− 5* 12 +/− 4* ND 28 +/− 6#ND = none detectedTotal Erythroid = CFU-E + BFU-ETotal CFC = Total Erythroid + CFU-GM + CFU-GEMM$p < 0.01*p < 0.005#p < 0.001**p < 0.0005

The number and distribution of colonies detected in the vehicle andendotoxin controls were no different from the standard control(containing no compound and no vehicle buffer). A dose-dependent toxiceffect (inhibitory effect on erythroid and myeloid progenitorproliferation) was seen in cultures incubated with 5-fluorouracil.Complete inhibition of growth was seen at the highest concentration of 5ug/ml. A significant decrease from control numbers was also seen at 1ug/ml for erythoid and myeloid growth and at 0.5 ug/ml for myeloidgrowth only, indicating that 5-FU is more toxic to the myeloid celllineage. Colony numbers recovered to near control levels at 0.1 ug/ml.

In the presence of each hIFN polypeptide or PEGASYS®, the morphology ofthe resulting erythroid and myeloid colonies was not perturbed. However,where colony numbers were affected so was the size of both colony types.All hIFN polypeptides and PEGASYS® were found to be toxic to the totalnumber of colony forming cells (myeloid and erythroid progenitors) atall concentrations tested, showing a slight dose dependent effect. hIFNpolypeptides and PEGASYS® did cause a slight gradual dose dependenteffect on progenitor compounds. PEGASYS® was found not to besignificantly toxic at 0.225 ug/ml, and 6HisM16pAF-30K PEG and6HisN65pAF-30K PEG were found not to be significantly toxic to erythoidprogenitors at 0.45 (6HisM16pAF-30K PEG only) and 0.225 ug/ml. However,for all compounds the total number of CFC was significantly inhibited atall concentrations tested. Colony size was also found to be smaller inthe presence of all compounds and size reflected the toxicity seen. Forexample, colonies in the presence of 3.6 ug/ml PEGASYS® were smallerthan those observed at 0.225 ug/ml.

Further analysis of the CFU-GM colony count (Y-axis) is shown as FIG.10, with International Units of anti-viral activity on the X-axis. Thespecific activity of PEGASYS® in anti-viral assays is 1 ng/unit. Massconcentrations of the PEGylated hIFN polypeptides were converted toInternational Units using the anti-viral IC50 values measured in the VSVreplication assay. The CFU-GM colony count for the control standard isplotted on the far right of the graph. CFU-GM colony counts for the fiveconcentrations of PEGASYS® tested in the assay are plotted. There is anincrease in colony count as PEGASYS® concentration is decreased fromapproximately 3600 units/ml to 225 units/ml. The colony counts of thePEGylated hIFN polypeptides are also shown.

All hIFN polypeptides showed a concentration dependent effect on theCFU-GM count. On a per unit basis, hIFN polypeptides 6HisI24pAF-30K PEGand 6HisN65pAF-30K PEG appeared to be more toxic than PEGASYS® in thisassay. 6HisN65pAF-30K PEG was at least 5-fold less potent than PEGASYS®in anti-viral assays, but was found to bind tightly to the IFNR2receptor. In contrast, 6HisE78pAF-30K PEG appeared to be approximatelytwo fold less toxic having equal colony counts as PEGASYS® at 2 foldhigher unit concentrations. For example, the 6HisE78pAF-30K PEG colonycount of 22 at a concentration of 1600 units/ml is compared to thePEGASYS® colony count of 21 at 900 units/ml. Further studies of toxicityinclude but are not limited to CFU-GM toxicity studies with 12 pointconcentration curves run from 15000 to 1 unit/ml for each hIFNpolypeptide to determine the IC50 for CFU-GM toxicity.

Table 7 shows hematopoietic toxicity data obtained with PEGASYS® andRibavirin. PEGASYS® was in a buffer of 20 mM Na Acetate, 137 mM NaCl,0.005% polysorbate 80, pH6.0 and had a starting concentration of 180ug/ml. Ribavirin (Calbiochem cat #555580) in PBS was at a startingconcentration of 7.63 mg/ml. Dilutions of each were tested, as shown inTable 7. TABLE 7 Total CFU- Total CFU-E BFU-E Erythroid CFU-GM GEMM CFCStandard 13 +/− 5 55 +/− 6  68 +/− 11 88 +/− 5 ND 156 +/− 7 PBS control16 +/− 7 57 +/− 4  73 +/− 10 85 +/− 6 ND  158 +/− 16 ABX buffer 13 +/− 360 +/− 4 73 +/− 7 91 +/− 5 ND  164 +/− 11 control PEGASYS ®  13.5 ug/mLND ND ND ND ND ND  4.5 ug/mL 11 +/− 3  2 +/− 1 13 +/− 3 10 +/− 3 ND  23+/− 5  1.5 ug/mL 15 +/− 2 11 +/− 3 26 +/− 4 18 +/− 5 ND  44 +/− 9  0.5ug/mL 17 +/− 3 28 +/− 3 45 +/− 4 32 +/− 7 ND  77 +/− 10  0.17 ug/mL 14+/− 4 25 +/− 5 39 +/− 2 46 +/− 3 ND  85 +/− 2 0.056 ug/mL 15 +/− 1 42+/− 8 57 +/− 9  49 +/− 10 ND 106 +/− 9 0.019 ug/mL 17 +/− 1 41 +/− 7 58+/− 7 63 +/− 8 ND 121 +/− 2 0.006 ug/mL 17 +/− 4 42 +/− 3 59 +/− 4 68+/− 6 ND 127 +/− 3 0.002 ug/mL 12 +/− 2 46 +/− 7 58 +/− 6 71 +/− 6 ND129 +/− 6 Ribavirin   636 ug/mL ND ND ND ND ND ND   212 ug/mL ND ND NDND ND ND 70.67 ug/mL ND ND ND ND ND ND 23.56 ug/mL ND ND ND ND ND ND 7.85 ug/mL ND ND ND ND ND ND  2.62 ug/mL ND ND ND ND ND ND  0.87 ug/mL19 +/− 1 31 +/− 5 50 +/− 5 42 +/− 6 ND  92 +/− 2  0.29 ug/mL 16 +/− 4 33+/− 3 49 +/− 2 69 +/− 5 ND 118 +/− 3  0.1 ug/mL 17 +/− 4 39 +/− 2 56 +/−6 73 +/− 4 ND 129 +/− 9  0.03 ug/mL 16 +/− 4 50 +/− 8 66 +/− 4 86 +/− 4ND 152 +/− 8  0.01 ug/mL 12 +/− 2 51 +/− 4 63 +/− 4  84 +/− 12 ND 147+/− 9 0.0036 ug/mL  16 +/− 1 53 +/− 7 69 +/− 7 83 +/− 9 ND  152 +/− 11Graphs generated that plot BFU-E, CFU-GM, or Total CFC vs. concentration(ug/ml) for each of the compounds are shown as FIGS. 12-17.Anti-Viral Assays

Antiviral activity of hIFN polypeptides may be measured by a variety ofassays. One such assay involves studying the reduction of cytopathiceffect (CPE) of cells infected with Vesicular Stomatitis Virus (VSV).VSV (ATCC) is produced by infecting the baby hamster kidney cell lineBHK21 (ATCC). Briefly, cells are infected with various amounts of virus,and 24 hours post infection the supernatant containing virus iscollected. Cell debris is eliminated by centifugation of the supernatantfor 5 minutes at 1,200 rpm. The stock of virus is stored in 1 mlaliquots at −80° C. The titer of virus is calculated by using plaqueassays. Titration is expressed by PFU/ml (plaque-forming unit).Additional assays for testing antiviral activity of hIFN polypeptides ofthe invention include, but are not limited to, an assay based on the HCVreplicon (Dr. Brent Korba at Department of Microbiology and Immunology,Georgetown University Medical Center). A variety of hIFN polypeptidesare assayed in the stably HCV RNA-replicating cell line, AVA5; this cellline is derivative of the human hepatoblastoma Huh7. Intracellular HCVRNA levels are measured 24 hours after the last dose of hIFN polypeptidehas been added. The RNA levels are measured using standard blothybridization techniques. Two strains of HCV replicon are tested, HCVstrain 1a and 1b. Another HCV assay is based upon measurement of HCVreplication in specific human cells that support HCV replication (Dr.Brent E. Korba or Dr. Thomas I. Michalak, M. D., Ph.D. (Faculty ofMedicine Health Science Centre Memorial University St. John's, Canada)).Another assay to assess the biological activity of hIFN polypeptidesutilizes woodchuck hepatocytes. Human IFN-α up-regulates class I MHCantigen expression and 2′-5-OAS mRNA in this type of cells (Dr. ThomasI. Michalak, M. D., Ph.D). The effects of hIFN polypeptides of theinvention on Class I MHC expression and/or 2′-5′-OAS levels are measuredin the hepatocytes.

To evaluate hIFN polypeptide activity, 3×10⁴ human WISH cells (ATCC)were seeded in a 96 well/plate and were subsequently infected with10,000 PFU of VSV per well. At the time of infection, different amountsof hIFN polypeptide were added. 48 hours post-infection the CPE wasevaluated; 42-48 hours was the minimum time required to obtain 100% CPE.CPE was identified by staining the cells with 0.1 ml of 1 mg/ml3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT)followed by spectrophotometric reading at 570 nm with 690 nm as thereference wavelength. MTT measures metabolic reduction in mitochondria.

Table 8 summarizes the IC50 values obtained with PEGASYS® andthirty-three hIFN polypeptides that have a substitution at the siteindicated and were PEGylated. UnPEGylated hIFN polypeptides with apara-acetylphenylalanine substitution were also tested with this assay.FIG. 9 shows dose response curves obtained with nine PEGylated hIFNpolypeptides and PEGASYS®. TABLE 8 hIFN IC₅₀ (ng/ml) 6His-L9-30K PEG11.7 6His-R12-30K PEG 17.7 6His-R13-30K PEG 27.9 6His-M16-30K PEG 3.86His-I24-30K PEG 1.1 6His-F27-30K PEG 3.5 6His-K31-30K PEG 3.86His-G37-30K PEG 1.8 6His-P39-30K PEG 10.3 6His-E41-30K PEG 3.56His-N45-30K PEG 1.9 6His-Q48-30K PEG 2.9 6His-K49-30K PEG 4.36His-Q61-30K PEG 50 6His-N65-30K PEG 50 6His-E78-30K PEG 1.76His-Y89-30K PEG 7.05 6His-E96-30K PEG 5.8 6His-I100-30K PEG 2.46His-G102-30K PEG 5.7 6His-V103-30K PEG 3.1 6His-T106-30K PEG 3.16His-E107-30K PEG 1.9 6His-P109-30K PEG 2.2 6His-L110-30K PEG 2.56His-E113-30K PEG 2.7 6His-L117-30K PEG 3.8 6His-R120-30K PEG 506His-R125-30K PEG 5.4 6His-K134-30K PEG 2.1 6His-R149-30K PEG 13.56His-E159-30K PEG 16.6 6His-K164-30K PEG 9.2 PEGASYS ® 7.6

Site specific PEGylation was found to alter the activity of IFN and mayseparate various biochemical properties of the protein. This effect ofsite specific PEGylation was found as a result of an analysis of theanti-viral IC50 data with the K_(D) of each hIFN polypeptide for thehigh affinity IFN receptor IFNR2, as shown in FIG. 11. The size of thebubbles represents the measured K_(D) of each hIFN polypeptide forIFNR2. Two hIFN polypeptides, 6HisI24pAF-30K PEG and 6HisE107pAF-30KPEG, were approximately five fold more potent in the cell basedanti-viral assay than PEGASYS®. Also, there was a lack of correlationbetween IFNR2 receptor K_(D) and anti-viral activity. Thus, there isevidence that site specific PEGylation can differentially alter thebiochemical activity of IFN. Thus, hIFN polypeptides have been producedthat have improved anti-viral potency that also differ in various othermeasured biochemical properties.

Example 26 Other Assays

Measurement of HLA-A, B, C Expression

An assay measuring the increase in cell surface MHC class I expressionin THP-1 cells may also be used to test activity of hIFN polypeptides.The human monocyte line THP-1 was cultured as indicated above. THP-1cells, at a cell concentration of 5×10⁵ cell/ml, were firstdifferentiated in the presence of 5 nM phorbol 12-myristate 13-acetate(Sigma, St Louis, Mo.). Differentiated THP-1 cells were then stimulatedwith increasing concentrations of IFNα2A for 24 hours at 37° C. IFNα2Aactivated cells were then harvested; washed and stained for surface HLAexpression. Immunofluorescent staining with APC-conjugatedanti-HLA-A,B,C antibody in the presence of propidium iodide (BDBiosciences, San Diego, Calif.) enabled sample analysis on the FACSArray with acquired data analysis on the Flowjo software. EC₅₀ valueswere estimated from dose response curves plotted with mean fluorescentintensity against protein concentration utilizing SigmaPlot. See FIG. 7.

PGE2 and cAMP Assays

Examples of in vitro assays that are used to evaluate hIFN polypeptidesinclude, but are not limited to, assays that detect an increase inprostaglandin E2 (PGE2) or decrease in cAMP as described for example, inWang et al, J. of Neuroimmunology 2004 156:107-112 and Jiang et al,Neurochemistry 2000 36:193-196, which are incorporated herein byreference. Additional citations describing PGE2 secretion assays includebut are not limited to, Mazzucco et al. J. Leukoc Biol 1996;60(2):271-7; Hoozemans et al. Exp. Gerontol. 2001 March; 36(3):559-70;Fiebich et al. J. Neurochem. 2000 November; 75(5):2020-8; Schwende etal. J Leukoc Biol 1996; 59(4):555-61; and Hoozemans et al. ActaNeuropathol (Berl.) 2001 Jan.; 101(1):2-8, which are incorporated hereinby reference. Modifications to these methods are known to one ofordinary skill in the art.

Cell based assays are performed, for example, comparing hIFNpolypeptides of the invention with controls that modulate PGE2 or cAMPlevels or current IFN therapeutics. Such assays measuring PGE2 secretioninvolve cell lines such as human THP-1 leukemia cells and humanneuroblastoma cells (SK—N—SH). Differentiated THP-1 cells challengedwith lipopolysaccharide (LPS) showed an increase in secretion of PGE2.In SK—N—SH cells, IL-ID increased PGE2 secretion. Such assays may beused to investigate the correlation of fever induction and/or theanalgesic effect found with current IFN therapies with modulatedsecretion of prostaglandin E2 (PGE2) or cAMP levels.

cAMP levels are detected as follows. 5×10⁴ SK—N—SH neuroblastoma cells(ATCC) are allowed to adhere overnight onto a 96-well assay plate(Applied Biosystems, Foster City, Calif.). Cells are then stimulated at37° C. for 15 minutes with (a) 10 uM forskolin alone; (b) 10 uMforskolin plus hIFN polypeptides (60 pM for non-PEG version and 600 pMfor PEG form) and (c) 10 uM forskolin plus hIFN polypeptides plus 20 uMnaloxone (an opioid receptor antagonist). Cells are then lysed, and thelysates are incubated with a cAMP-alkaline phosphatase conjugate and ananti-cAMP antibody for 1 hour at room temperature. A chemiluminescentsubstrate is then added and the level of light emission measured on aTopcount luminescent reader. The amount of cAMP present in the lysatesis quantitated from a cAMP standard curve.

Gene Expression Profiling

Transcriptional activity of monoPEGylated interferon-α-2a isomers isdescribed in Foser et al. Pharmacogenomics Journal 2003; 3:312-319 usingoligonucleotide array transcript analysis. Cell lines that may be usedin expression studies include but are not limited to melanoma cell linessuch as ME15. Alternate assays to DNA arrays may be performed with hIFNpolypeptides of the invention to provide mRNA profiling or differentialgene expression information.

Additional Assays

Assays to support preliminary formulation studies include assays forprotein de-amidation, aggregation, oxidation, acetylation, and otherstability indicating assays. Other assays include, but are not limitedto, assays that investigate other potential degradation products,including but not limited to, disulfide rearrangements and proteolyticdegradation products.

Example 27

This example describes an alternate system for cloning, expression andpurification of hIFN polypeptides of the invention. Possible advantagesof this alternate system include, but are not limited to, the deletionof one or more steps that involve refolding of hIFN polypeptides. NusAfusion proteins and TEV are discussed in Davis et al. Biotechnol Bioeng(1999); 65:382-388; Shih et al. Protein Science 2005; 14:936-941; andU.S. Pat. Nos. 5,162,601, 5,532,142; 5,766,885; and 5,989,868, all ofwhich are incorporated by reference herein.

Cloning and Constructs for Nus-hIFN Polypeptide Fusions

To generate NusA-hIFN fusions, hIFN nucleotide sequences were cloneddownstream of NusA between SpeI and KpnI restriction sites, separated bythe sequence GSGENLYFQ (which includes a GSG linker and the recognitionsequence for TEV). All initial cloning steps were completed usingNovagen's pET44 vector, and then the whole Nus-IFN fusion cassette wastransferred into another vector that allows for expression of hIFNcontrolled by the T7lac promoter and induction of protein expressionwith 1 mM IPTG. The transformation of E. coli (Origami 2) withconstructs containing the modified hIFN polynucleotide sequence and theorthogonal aminoacyl tRNA synthetase/tRNA pair (specific for the desirednon-naturally encoded amino acid) allows the site-specific incorporationof non-naturally encoded amino acid into the hIFN polypeptide.

Purification and Cleavage of Nus-hIFN Fusions

A 5 ml LB culture was grown from a single colony of E. coli transformedwith a construct encoding the NusA-hIFN fusion. 0.5 liters of LB withampicillin was inoculated, and the non-naturally encoded amino acidp-acetyl-phenylalanine was added to the culture. The culture was grownovernight at 28° C.-32° C. When the OD of the culture was greater than0.8-1, the culture was transferred to 30° C., and induction wasperformed with 1 mM IPTG. The culture continued to grow for 4 hours at30° C., and a sample was collected for SDS-PAGE analysis. The cells werecollected by centrifugation and frozen at −80° C.

The cells were lysed in 25-30 ml of 50 mM Tris, pH 8, 200 mM NaCl, 5%glycerol. The samples were sonicated in a glass beaker for 25 sec 5-6times, while keeping the samples on ice. The cells were incubated with10 uL DNAse for 30 minutes at room temperature. The samples were thenspun down and the supernatant removed. 5 mM imidazole was added to thesupernatant.

The supernatant was then loaded onto a 5 ml HisTrap column at 3 ml/min.Prior to loading the supernatant, the HisTrap column is equilibratedwith 20 mM Tris pH 8, 300 mM NaCl, 0.005% Tween80, 10 mM imidazole(Buffer A). After washing the column with Buffer A, elution wasperformed with the following step gradient: 4 column volumes of 8%Buffer B (20 mM Tris pH 8, 300 mM NaCl, 0.005% Tween80, 0.5M imidazole);5 column volumes of 30% Buffer B; and 100% Buffer B wash. The NusA-hIFNfusion eluted during the 30% Buffer B step. The column was washed with1M NaOH/1M NaCl and was stripped and recharged (or replaced) after fiveruns.

Samples from the step gradient were analyzed by SDS-PAGE, and thefractions with the fusion (ca 75 kDa) were pooled and dialyzed against20 mM Tris, pH 8, 20 mM NaCl, 5% glycerol overnight at 4° C. The samplewas diluted to 30 uM protein with dialysis buffer. The following wasadded to the sample: cysteamine to 0.2 mM, EDTA to 1 mM, and TEV to 0.8uM. The samples were then incubated for 24-48 hours at 4° C., orovernight at room temperature.

After the incubation, the samples were diluted 1:3 with ddH₂O and loadedonto a 5 ml Q HP column. Elution was performed with a 0-40% gradient of10 mM Tris, pH 8 (Buffer A′) into Buffer B′ (10 mM Tris, pH 8, 1M NaCl)over eighteen column volumes. hIFN polypeptides eluted at approximately100 mM NaCl; TEV at 250 mM NaCl, and NusA at 400 mM NaCl. The column waswashed with 2.5 mM NaCl, followed by 1M NaCl/1M NaOH.

Eluted fractions were analyzed by SDS-PAGE. hIFN polypeptide fractionswere pooled, and the pH of the pool was adjusted by adding 1/20 volumeof 1M NaAc, pH 4.5, and the hIFN pool was then dialyzed against 20 mMNaAc, pH 4, 20 mM NaCl, 5% glycerol overnight. The sample was thenconcentrated to greater than 1 mg/ml, keeping the volume greater than300 mcl. The extinction coefficient at 280 nm is 22,500 M⁻¹ or 1.17mg/ml⁻¹.

PEGylation

Oxyamino-derivatized 30K PEG was added to hIFN polypeptide at a 1:12molar ratio (30 ng/mg IFN/ml), and the mixture was incubated at 28° C.for 16-48 hours. FIG. 19 shows the 30K PEG used in the conjugation. Thesample was diluted ten times with SP buffer A (50 mM Na acetate, pH 5).To purify the PEGylated hIFN polypeptides, a 5 ml SP HP AKTA column wasused with a gradient of 0-50% SP buffer B (50 mM Na acetate, pH 5, 0.5 MNaCl, 10% ethylene glycol) over ten column volumes. PEGylated IFN elutedaround 100 mM salt, followed by non-PEGylated monomer (baselineseparation). The fractions of PEGylated hIFN were pooled, dialyzedagainst the following storage buffer: 20 mM NaAcetate, pH 6, 0.005%Tween, 125 mM NaCl. The samples were then concentrated to >8 microM andstored at 4° C.

Purification of TEV

Purified TEV was generated with the following protocol. 0.5-2 litercultures of BL21(DE3)RIL cells (Stratagene) were transformed withpRK793-TEV(S219V) plasmid (from NCI) at 30° C. Induction was performedwhen the OD600 was 0.8-1.2, and growth was continued for 4-5 hours at30° C. The cells were resuspended in 20-30 ml of NTA buffer (50 mM Tris,pH 7, 300 mM NaCl). The samples were then sonicated five times forthirty seconds on ice, incubated at room temperature for thirty minutes,and centrifuged for twenty minutes at 12,000 rpm. The supernatant wasremoved, and imidazole was added to 15 mM.

The supernatant was loaded onto a 5 mL HisTrap column equilibrated inNTA buffer supplemented with 15 mM imidazole. Elution was performed witha gradient of 0-100% B (NTA buffer with 0.5M imidazole) over eighteencolumn volumes. TEV eluted as a broad peak starting 200 mM imidazole.The eluted fractions were analyzed by SDS-PAGE, and the fractionscontaining clean TEV were pooled. The pooled fractions were dialyzedovernight against the following buffer (30 mM Tris, pH 7.2, 200 mM NaCl,0.5 mM EDTA, 1 mM DTT). The dialyzed sample was centrifuged to removeany precipitate.

The spun material was then dialyzed four-fold with ddH₂O and loaded onto5 ml SP FF AKTA column equilibrated with Buffer A (10 mM Tris pH 7, 50mM NaCl). The column was eluted with a linear gradient 0-100% B (10 mMTris, pH 7, 0.5M NaCl) over eighteen column volumes. The eluted TEVfractions were pooled, and the following was added to the pool: 1 mMDTT, 1 mM EDTA, 20% glycerol. The sample was concentrated to >20 uM(ext. coeff. 32,500, pI is 9.6), aliquoted at 500 mcl/tube, and storedat −20° C.

Anti-Viral Activity

To evaluate the activity of hIFN polypeptides generated via the methodsdescribed in this Example, 3×10⁴ human WISH cells (ATCC) were seeded ina 96 well/plate and were subsequently infected with 10,000 PFU of VSVper well. At the time of infection, different amounts of hIFNpolypeptide were added. 48 hours post-infection the CPE was evaluated;42-48 hours was the minimum time required to obtain 100% CPE. CPE wasidentified by staining the cells with 0.1 ml of 1 mg/ml3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT)followed by spectrophotometric reading at 570 nm with 690 nm as thereference wavelength. MTT measures metabolic reduction in mitochondria.The antiviral activity of five PEGylated hIFN polypeptides and PEGASYS®is shown in FIG. 18. Methionyl hIFN polypeptides comprising anon-naturally encoded amino acid are designated with an “M-” hIFNpolypeptides without a methionine as the first amino acid are alsogenerated using methods involving NusA.

Example 28

This example describes methods to measure in vitro and in vivo activityof hIFN polypeptides of the invention.

Cell Binding Assays.

Cells (3×10⁶) are incubated in duplicate in PBS/1% BSA (100 μl) in theabsence or presence of various concentrations (volume: 10 μl) ofunlabeled IFN, hIFN or GM-CSF and in the presence of ¹²⁵I-IFN (approx100,000 cpm or 1 ng) at 0° C. for 90 minutes (total volume: 120 μl).Cells are then resuspended and layered over 200 μl ice cold FCS in a 350μl plastic centrifuge tube and centrifuged (1000 g; 1 minute). Thepellet is collected by cutting off the end of the tube and pellet andsupernatant counted separately in a gamma counter (Packard).

Specific binding (cpm) is determined as total binding in the absence ofa competitor (mean of duplicates) minus binding (cpm) in the presence of100-fold excess of unlabeled IFN (non-specific binding). Thenon-specific binding is measured for each of the cell types used.Experiments are run on separate days using the same preparation of¹²⁵I-IFN and should display internal consistency. ¹²⁵I-IFN demonstratesbinding to the Daudi cells. The binding is inhibited in a dose dependentmanner by unlabeled natural IFN or hIFN, but not by GM-CSF or othernegative control. The ability of hIFN to compete for the binding ofnatural ¹²⁵I-IFN, similar to natural IFN, suggests that the receptorsrecognize both forms equally well.

In Vivo Studies from PEGylated IFN

PEG-hIFN, unmodified hIFN and buffer solution are administered to miceor rats. The results will show superior activity and prolonged half lifeof the PEGylated hIFN of the present invention compared to unmodifiedhIFN which is indicated by significantly increased inhibition of viralreplication using the same dose per mouse or rat.

Measurement of the In Vivo Half-Life of Conjugated and Non-ConjugatedhIFN and Variants Thereof.

Male Sprague Dawley rats (about 7 weeks old) are used. On the day ofadministration, the weight of each animal is measured. 100 pg per kgbody weight of the non-conjugated and conjugated hIFN samples are eachinjected intravenously into the tail vein of three rats. At 1 minute, 30minutes, 1, 2, 4, 6, and 24 hours after the injection, 500 μl of bloodis withdrawn from each rat while under CO₂-anesthesia. The serum isisolated from the blood samples by centrifugation (4° C., 18000×g for 5minutes). The serum samples are stored at −80° C. until the day ofanalysis. The amount of active IFN in the serum samples is quantified bythe IFN in vitro activity assay after thawing the samples on ice.

Antiviral Activity

There are many assays known to those skilled in the art that measure thedegree of resistance of cells to viruses (McNeill T A, J ImmunolMethods. (1981) 46(2):121-7). These assays generally can be categorizedinto three types: inhibition of cytopathic effect; virus plaqueformation; and reduction of virus yield. Viral cytopathic effect assaysmeasure the degree of protection induced in cell cultures pretreatedwith IFN and subsequently infected with viruses. Vesicular stomatitisvirus, for instance, is an appropriate virus for use in such an assay.This type of assay is convenient for screening numerous different IFNs,as it can be performed in 96-well plates. An example of a cytopathiceffect reduction assay is detailed in Wang et al. NeuroReport (2001)12(4):857-859. Plaque-reduction assays measure the resistance ofIFN-treated cell cultures to a plaque-forming virus (for instance,measles virus). One benefit to this assay is that it allows precisemeasurement of a 50% reduction in plaque formation. Finally, virus yieldassays measure the amount of virus released from cells during, forinstance, a single growth cycle. Such assays are useful for testing theantiviral activity of IFNs against viruses that do not cause cytopathiceffects, or that do not build plaques in target-cell cultures. Themultiplicity of infection (moi) is an important factor to consider whenusing either plaque-reduction or virus-yield assays.

Other clinically important interferon characteristics are also easilyassayed in the laboratory setting. One such characteristic is theability of an interferon polypeptide to bind to specific cell-surfacereceptors. For instance, some IFNα-2 as exhibit different cell-surfaceproperties compared to IFNα-2b, the IFN most widely used in clinicaltrials. While IFNα-2b is an effective antiviral agent, it causessignificant adverse side effects. Interferons that exhibit distinctbinding properties from IFNα-2b may not cause the same adverse effects.Therefore, interferons that compete poorly with IFNα-2b for bindingsites on cells are of clinical interest. Competitive interferon bindingassays are well known in the art (Hu et al., J Biol Chem. (1993) June15; 268(17):12591-5; Di Marco et al., (1994) Biochem. Biophys. Res.Comm. 202:1445-1451, which are incorporated by reference herein). Ingeneral, such assays involve incubation of cell culture cells with amixture of ¹²⁵I-labeled IFNα-2b and an unlabeled interferon of interest.Unbound interferon is then removed, and the amount of bound label (andby extension, bound ¹²⁵I-labeled IFNα-2b) is measured. By comparing theamount of label that binds to cells in the presence or absence ofcompeting interferons, relative binding affinities can be calculated.

Another prominent effect of IFNα's is their ability to inhibit cellgrowth, which is of major importance in determining anti-tumor action.Growth inhibition assays are well established, and usually depend oncell counts or uptake of tritiated thymidine ([³H] thymidine) or anotherradiolabel. The human lymphoblastoid Daudi cell line has proven to beextremely sensitive to IFNα's, and it has been used to measureantiproliferative activity in many IFNα's and derived hybridpolypeptides (Meister et al., J Gen Virol. (1986) August; 67 (Pt8):1633-43). Use of this cell line has been facilitated by its abilityto be grown in suspension cultures (Evinger and Pestka, (1981) MethodsEnzymol. 79:362-368). IFNα's also exhibit many immunomodulatoryactivities (Zoon et al., (1986) In, The Biology of the InterferonSystem. Cantell and Schellenkens, Eds., Martinus Nyhoff Publishers,Amsterdam).

Although IFNs were first discovered by virologists, their first clinicaluse (in 1979) was as therapeutic agents for myeloma (Joshua et al.,(1997) Blood Rev. 11(4):191-200). IFNα's have since been shown to beefficacious against a myriad of diseases of viral, malignant,angiogenic, allergic, inflammatory, and fibrotic origin (Tilg, (1997)Gastroenterology. 112(3):1017-1021). It has also proven efficacious inthe treatment of metastatic renal carcinoma and chronic myeloid leukemia(Williams and Linch, (1997) Br. J. Hosp. Med. 57(9):436-439). Clinicaluses of IFNs are reviewed in Gresser (1997) J. Leukoc. Biol.61(5):567-574 and Pfeffer (1997) Semin. Oncol. 24(3 Suppl.9):S9-S63S969.

Analgesia Assays

Pain threshold studies may be performed with hIFN polypeptides toinvestigate the analgesic effect of these compounds. One such analgesiaassay is described in Wang et al. NeuroReport (2001) 12(4):857-859,which is incorporated by reference herein.

Example 29

This example describes methods to measure in vivo activity and toxicityof hIFN polypeptides of the invention.

Animal models used to study antiviral activity of hIFN polypeptidesinclude, but are not limited to, the chimpanzee HCV (Purcell R H, FEMSMicrobiol Rev. 1994 July; 14(3):181-91), the HCV-Trimera mouse (Ilan Eet al. J Infect Dis. 2002 Jan. 15; 185(2):153-61), and the Alb-uPAtransgenic mouse models (Mercer D F et al. Nat Med. 2001 August;7(8):927-33; Kneteman, N et al (2003) 10th HCV Meeting Kyoto Japan,P-187), all of which are incorporated by reference herein. Woodchuckhepatitis virus (WHV) infection in woodchucks is a useful model to studythe pathogenesis, prevention, and treatment of HBV infection (Berraondo,P. et al, J. Med. Virology 2002; 68, 424-432). The condition ofwoodchucks exposed prenatally to WHV is similar to HBV infection.Compounds, either PEGylated human interferon polypeptides, PEGylatedwoodchuck interferon compound analogs, or PEGylated human interferonpolypeptides modified in some way so as to increase their speciescross-reactivity towards woodchuck sufficiently for antiviral efficacyevaluation, are evaluated in the woodchuck hepatitis model.

Cynomolgus monkeys are also used to study in vivo activity and bonemarrow toxicity. Activity is assayed by measuring the induction ofdownstream markers including but not limited to 2′,5′-OAS by hIFNpolypeptides of the invention compared to current IFN therapeutics suchas PEGASYS®. Circulating blood cells, including but not limited toneutrophils, RBCs, and platelets are evaluated in bone marrow toxicitystudies after administration of hIFN polypeptides compared to currentIFN therapeutics such as PEGASYS®. 2′,5′-OAS is an enzyme induced byinterferon and is directly related to the anti-viral activity ofinterferon. This enzyme produces a mixture of oligonucleotides (2-5A)which are thought to activate the inactive RNAse present in cells andhinder the protein synthesis of cells or viruses through the breakdownof mRNA. Detection of 2′,5′-OAS activity may be performed by a varietyof assays known to those of ordinary skill in the art, including but notlimited to, radioimmunoassay (RIA; EIKEN) which measures levels of 2-5A.Cynomolgus serum, for example, may be used to measure 2′,5′-OAS activitylevels. Neopterin is a non-species specific immune marker that isproduced by macrophages upon exposure to interferon. Detection ofneopterin may be performed by a variety of assays known to those ofordinary skill in the art, including but not limited to, EIA (IBL).Levels of other molecules may be measured in such studies, including butnot limited to cortisol levels. Cortisol, an element of the HPA axis,has been implicated in depression, stress, and weight gain. Detection ofcortisol may be performed by a variety of assays known to those ofordinary skill in the art, including but not limited to, EIA (RnD).Cytokine panels may be performed throughout the study afteradministration of hIFN polypeptides of the invention and levels ofvarious cytokines compared to basal levels to evaluate toxicity of thehIFN polypeptides. A variety of assays known to those of ordinary skillin the art may be used, including but not limited to, ELISA, bead arrays(BD Biosciences) and other assay formats (Luminex) and may involvedifferent types of readouts, including but not limited to, fluorescentreadouts. Measurement of compound levels in serum or plasma may bedetected using a variety of assays known to those of ordinary skill inthe art, including but not limited to, ELISA (PBL). Antibodies used fordetection by ELISA or other methods may be anti-IFN alpha antibodies oranti-PEG antibodies for hIFN polypeptides that are PEGylated. Variousassay formats known to those of ordinary skill in the art may be usedfor detection including those involving fluorescence.

Evaluation of hIFN polypeptides of the invention is performed with thefollowing pre-clinical plan. In Study 1, a dose-range findingpharmacokinetics (PK), pharmacodynamics (PD), and safety study isinitiated in female cynomolgus monkeys and involves placebo and fourdoses of PEGASYS® (0, 1.5, 15, 50, and 150 ug/kg). The monkeys areacclimated for two weeks. 15, 50 and 150 ug/kg of PEGASYS® isadministered on Day 1 and Day 5. 1.5 ug/kg of PEGASYS® is administeredon Day 1 only. There are three female monkeys in each of the fivegroups. The duration of in-life is 14 days with an option to proceed forat least an additional week in the event that data so warrant. Theendpoints are compound levels in the plasma measured by integrating theAUC through Day 8 and Day 15; and 2′,5′-OAS and neopterin levels throughDay 15. In this study, hematology studies are performed investigatingthe effect of the administered hIFN polypeptides on neutrophils andplatelets on Day 3, Day 8 and Day 15. A prebleed is also done on theanimals for comparison. Cytokine panels are also performed through Day15, measuring levels of various cytokines including but not limited to,IL-2, IL-4, IL-5, IL-6, TNF, and IFN-γ. Cortisol is measured as wellthrough Day 15. Rectal body temperature of the monkeys is also bemeasured, since fever is also a known side effect of current IFNtherapeutics.

After one or two doses have been selected from the first study, Study 2involves comparing hIFN polypeptides of the invention with PEGASYS® atthe one or two doses. If a single dose is suitable for a comparison ofPK, efficacy, and toxicity, then the particular dose of PEGASYS® istested in cynomolgus monkeys and compared to one or more hIFNpolypeptides at the same dose, and placebo. Three female monkeys are ineach group; the acclimation period is two weeks. As in Study 1,2′,5′-OAS levels, hematology, body temperature, cortisol, and cytokinepanels are studied. If two doses are required i.e. a low dose isrequired for PK and efficacy and another dose is needed for toxicitycomparison, then PEGASYS® is compared to one or more hIFN polypeptidesat both doses and placebo. As in Study 1, 2′,5′-OAS levels, hematology,body temperature, cortisol, and cytokine panels are studied. The end ofstudy in-life is Day 15, with an option to proceed for at least anadditional week in the event that data so warrant.

Bone marrow is collected and evaluated from animals exposed to hIFNpolypeptides of the invention. For example, bone marrow aspirates and/orcore biopsies from treated animals (e.g. from primates) are examinedhistologically, and the ratio of myeloid to nucleated erythroid cells(M:E ratio) is estimated. A low M:E ratio, in conjunction withneutropenia, may be indicative of granulocytic hypoplasia with decreasedproduction of neutrophils. Isolated bone marrow cells from treatedanimals are assayed for hematopoietic progenitors. That is, cellisolates are subjected to CFU-GM or BFU-E colony assay. The results(i.e. number of hematopoietic progenitor cells) would show the effect,if any, of treatment on myeloid and/or erythroid progenitor cells.

Such bone marrow toxicity studies may involve hIFN polypeptidesadministered via different routes, including but not limited to,subcutaneously and intravenously. Intravenous administration may allowfor longer dosing regimens and better evaluation of bone marrow toxicitythan subcutaneous administration. Such improvements may result from amodulation of immunogenicity or an antibody response to the compoundadministered. Toxicity studies include evaluation of neutrophil andplatelet number/mL of plasma or serum.

Other animal models include but are not limited to, depression models.One mouse model involves the Tail-Suspension Test. Assays measuring thelevels of cortisol and ACTH in mice after administration of hIFNpolypeptide are also performed. Both cortisol and ACTH are components ofthe HPA axis and have been implicated in playing a role ininterferon-treatment induced depression in patients.

Example 30

This example details the isolation of hIFN polypeptides and themeasurement of hIFN activity and affinity of these hIFN polypeptides forthe hIFN receptor.

Purification by Copper Column

500 ml LB Shake Flask Expression:

For each hIFN polypeptide, E. coli transformed with a construct encodingan orthogonal tRNA (J17 described in U.S. Patent Publication No.20030108885) and an orthogonal aminoacyl tRNA synthetase (E9 describedin U.S. patent application Ser. No. 11/292,903 entitled “Compositions ofAminoacyl-tRNA Synthetase and Uses Thereof”) with a mutated hIFNpolynucleotide were streaked onto a plate to obtain colonies. 2 mL LBcontaining antibiotic was inoculated with a single colony from thefreshly transformed plate. The culture was incubated under constantagitation (250 rpm) at 37° C. until the culture reached an OD600 ofapproximately 0.05-0.3 (approximately 5-6 hours). 500 mL LB (containingantibiotic and 4 mM pAF) was inoculated with all or part of the 2 mLculture, normalizing the inoculation to synchronize cultures. Theculture was incubated under constant agitation (250 rpm) at 37° C. untilculture reached an OD600 of approximately 1.0 (approximately 4-5 hours).The culture was then induced with 1 mM IPTG, and the culture wasincubated overnight at 37° C. The cells were harvested by centrifugationat 8000 rpm/12000 g; 15 minutes; 4° C. The cell paste was scraped outand stored in 40 mL Oak Ridge tubes. The cell pellets were then frozenat −80° C. until the inclusion body preparation was initiated.

Inclusion Body Preparation from 500 mL Shake Flask:

Buffer 1 was 50 mM NaAc pH 6.0; 100 mM NaCl; 1 mM EDTA; 1.0% TritonX-100. Buffer 2 was 50 mM NaAc pH 6.0; 100 mM NaCl; 1 mM EDTA. Table 9shows the procedure used for inclusion body preparation. TABLE 9 VolumeCentrifugation # Step Technique Buffer (mL) (g, minutes) 1 Lysis AvestinC3, 2 1 23 20000, 15 passes 2 Wash 1 Sonication, 30 1 25 20000, 15seconds 3 Wash 2 Sonication, 30 1 25 20000, 15 seconds 4 Rinse 1Sonication, 30 2 25 20000, 15 seconds 5 Rinse 2 Sonication, 30 2 2520000, 15 seconds

For the Lysis step, cells were resuspended in 23 mL chilled Buffer 1 byshaking for 1 hour at 4° C. The Avestin C3 was cleaned and rinsed with100 mL water and then with 30 mL Buffer 1. The cells were thenhomogenized with 2 passes in Avestin at 15,000-20,000 psi with coolingat 4° C. in the following manner: 1) The sample was added. 2) The samplewas processed once until Avestin reservoir was nearly empty; the samplewas collected in the original tube (also done for subsequent steps). 3)The collected sample was added back to the Avestin reservoir andre-homogenized. 4) The sample was washed out by homogenizing 12 mLBuffer 1; this was added to the sample. 5) The sample was transferred to40 mL Oak Ridge Centrifuge tube. 6) The sample was stored and 100 mLwater was run through the Avestin, adding it slowly while running toflush the system. 7) 30 mL Buffer 1 was then added to the reservoir andhomogenized to prepare for next sample. 8) The steps were then repeatedto process the next sample.

For the Wash and Rinse steps, a spatula was used to loosen the pelletoff the tube after pouring off supernatant from the last wash andpouring in fresh buffer. Sonication was performed at 75% power with anormal tip, not a microtip. The tip was rinsed with water and wiped witha Kimwipe between samples.

During the first 4 steps, the samples were spun in 40 mL Oak Ridge tubesused to store cell pellets. Rinse 2 was spun by splitting each sample to2 15 mL conical tubes.

Solubilization and Refolding:

Solubilization was performed with 8M GndHCl pH between 5.5-8.5 using 50mM NaAc or Tris. The inclusion bodies were solubilized to a finalconcentration between 5-10 mg/mL. The material was refolded or stored at−80° C. for long term storage.

Refolding was performed by diluting solubilized material to a finaltotal protein concentration of 0.5 mg/mL in 4° C. Refold Buffer (100 mMNaAc, pH 6.0 or 50 mM Tris, pH 8.0; 0.1% Tween 20). The refoldingmixture was stored at 4° C. for 24 to 72 hours.

Cu Purification:

The refolded sample was allowed to warm to room temperature. A finalconcentration of 50 μM CuCl was added to the refolded sample, and thesample was incubated at room temperature for 10-30 minutes. The samplewas filtered with a 0.22 μm PES filter and loaded onto a ChelatingSepharose HP column (GE Healthcare) charged with Cu and equilibratedwith Cu Buffer A (50 mM NaAc, pH 5.0; 150 mM NaCl; 0.1% Tween 20). Alinear gradient was run to 100% Cu Buffer B (100 mM NaAc, pH 3.5; 150 mMNaCl; 0.1% Tween 20) over 18 column volumes, and samples were collectedin tubes containing 1 mM EDTA.

SP HP Purification of Cu Pool:

The hIFN polypeptide peak was collected from the Cu column and loadedstraight onto a SP HP column (GE Healthcare) equilibrated in SP Buffer A(50 mM NaAc, pH 5.0; 1 mM EDTA). A linear gradient was run to 50% SPBuffer B (50 mM NaAc, pH 5.0; 0.5M NaCl; 10% ethylene glycol; 1 mM EDTA)over 15 column volumes.

PEGylation and Purification:

The hIFN polypeptide samples were pooled from the SP HP column andconcentrated to 1.0-2.0 mg/mL. 10% Acetic Acid was added to the sampleto drop the pH down to 4.0. Oxyamino-derivatized 30K PEG was added tohIFN polypeptide at a 1:12 molar ratio (30 mg/mg IFN/ml), and themixture was incubated at 28° C. for 48-72 hours. FIG. 19 shows the 30KPEG used in the conjugation. One hIFN polypeptide comprising thenon-natural amino acid para-acetylphenylalanine at position 107 wasconjugated to a branched 40K PEG. This PEG is shown in FIG. 20 ascompound IV (mPEG (40K) aminoethoxyamine hydrochloride), and the figureshows the synthesis scheme from mPEG (40K) p-nitrophenolcarbonate. Themolar ratio and conjugation conditions as well as the purificationmethod used for the 40K PEGylated product was the same as the 30KPEGylated products. The sample was diluted ten-fold with SP Buffer A (50mM Na acetate, pH 5; 1 mM EDTA). To purify the PEGylated hIFN, a SP HPcolumn (GE Healthcare) was used with a gradient of 0-50% SP buffer B (50mM Na acetate, pH 5, 0.5 M NaCl, 10% ethylene glycol; 1 mM EDTA) over 15column volumes. PEGylated IFN eluted around 100 mM salt, followed bynon-PEGylated monomer (baseline separation). The fractions of PEGylatedhIFN were pooled, dialyzed against the following storage buffer: 20 mMNaAcetate, pH 6, 0.005% Tween, 125 mM NaCl. Samples were concentrated to1.0-2.0 mg/mL and stored at −80° C.

The method used to functionalize the glycerol-based branched 40K PEGshown was as follows (FIG. 20). To a mixture of mPEG(40 K)p-nitrophenolcarbonate I (20 g, 0.5 mmol) and N-aminoethoxyphthalimidehydrochloride II (0.6 g, 2.5 mmol) in N,N-dimethylformamide-methylenechloride (1:2, 150 mL) were added diisopropylethylamine (DIEA, 0.27 mL,1.5 mmol) and 4-dimethylaminopyridine (DMAP, cat.). The resultantmixture was stirred at room temperature for 20 h. Ethyl acetate-hexanes(1:1, 1 L) was added. The precipitate was filtered, washed withethylacetate-hexanes (1 L) and dried in vacuo to afford product III as awhite powder.

mPEG(40K) N-aminoethoxyphthalimide III from the previous step wasdissolved in a 0.5 M solution of hydrazine in methylenechloride-methanol (1:1, 100 mL). The resultant mixture was stirred atroom temperature for 1.0 h and then washed with aqueous HCl solution(0.5 N, 300 mL). The organic layer was dried over anhydrous Na₂SO₄,filtered and concentrated. The residue was dissolved in CH₂Cl₂ (100 mL).Ethyl acetate-hexanes (1:1, 1 L) was added to precipitate the mPEG(40K)aminoethoxyamine hydrochloride product IV (17.6 g, 88% from I) as awhite powder.

Biacore Studies (Receptor Binding Affinity)

The sequence for the IFNAR2 extracellular domain (consisting of 206amino acids ending with sequence LLPPGQ) was amplified from cloneMHS1011-61064 (OpenBiosystems, Huntsville, Ala.). This insert was clonedinto the pET20 expression vector (Novagen) downstream of the T7promoter. Protein expression was induced with 0.4 mM IPTG in BL21(DE3)cells (Novagen).

Since the expressed protein was insoluble, the inclusion bodies werepurified from lysed cells and solubilized in 6M GndCl. A 5 ml aliquot(50 mg amount) was reduced with 10 mM DTT for 45 minutes at 37° C. Thenthe mixture was injected into 200 ml of refolding buffer which consistedof 50 mM Tris pH 8, 20 mM NaCl, 0.5 M Arginine, 10% glycerol at 4° C.and incubated overnight with gentle stirring.

The refolding reaction was then concentrated to 25 ml using an Amiconstirring cell, and dialyzed overnight against 20 mM Tris, pH 8, 20 mMNaCl, 10% glycerol. Monomeric refolded IFNAR ECD was purified on HP QSepharose using the AKTA FPLC system (Amersham). Purified IFNAR2 ECD wasimmobilized on CM5 Biacore chip using a lysine-specific couplingprocedure recommended by the manufacturer. About 200 RUs of functionalprotein were immobilized. Various concentrations of IFN variants inHBS-EP buffer (Biacore) were injected at a flow rate of 50 mcl/minuteover the flowcell containing immobilized IFNAR2, and a control flowcellcontaining immobilized bovine serum albumin. Sensograms generated werefit to the 1:1 interaction model to calculate k_(on), k_(off) and K_(d)values using BiaEvaluation software (Biacore). The receptor bindingaffinity of hIFN polypeptides PEGylated at the non-naturally encodedamino acid (para-acetylphenylalanine; pAF) were observed. Additionalmutants were tested that were PEGylated at pAF and had a naturallyencoded amino acid substitution at position 149. Methionyl wild-typeinterferon (M-WT) was included as a control sample. Table 10 shows thek_(on), k_(off), and K_(d) obtained. hIFN polypeptides shown do not havea 6-His tag and were refolded as described in this Example.

Table 10: Binding Parameters for IFNα2A: IFNAR2 Interaction, asDetermined by SPR k_(on), × 10⁻⁴ hIFN polypeptide 1/M * s k_(off), 1/sK_(d), nM M-WT 640 0.025 4 T6 pAF-30K PEG 8.3 0.045 540 M16 pAF-30K PEG6.6 0.043 650 T108 pAF-30K PEG 13 0.035 280 M111 pAF-30K PEG 10.5 0.046430 D114 pAF-30K PEG 10.4 0.040 380 E107 pAF-40K PEG 7.7 0.035 460 E107pAF-30K PEG 17 0.031 180 N45 pAF-30K PEG 8 0.048 600 Q46 pAF-30K PEG 8.10.038 460 F64 pAF-30K PEG 15 0.036 240 E78 pAF-30K PEG 9.7 0.033 335 E87pAF-30K PEG 5.9 0.026 440 Y85 pAF-30K PEG 11 0.054 490 Q101 pAF-30K PEG9 0.038 425 E96 pAF-30K PEG 13 0.038 300 A118 pAF-30K PEG 11 0.037 350N156 pAF-30K PEG 9.9 0.038 380 R149 pAF-30K PEG >10 uM R149Y/E107pAF-30K PEG >10 uM R149S/E107 pAF-30K PEG >10 uM R149E/E107 pAF-30KPEG >10 uM A145 pAF-30K PEG no binding observedAnti-Viral Assays

To evaluate hIFN polypeptide anti-viral activity, 3×10⁴ human WISH cells(ATCC) were seeded in a 96 well/plate and were subsequently infectedwith 10,000 PFU of VSV per well. At the time of infection, differentamounts of hIFN polypeptide were added. 48 hours post-infection the CPEwas evaluated; 42-48 hours was the minimum time required to obtain 100%CPE. CPE was identified by staining the cells with 0.1 ml of 1 mg/ml3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT)followed by spectrophotometric reading at 570 nm with 690 nm as thereference wavelength. MTT measures metabolic reduction in mitochondria.

Table 11 and 12 shows the % relative anti-viral activity based on theIC₅₀ values obtained with hIFN polypeptides that have a non-naturalamino substitution (para-acetylphenylalanine, pAF) at the site indicatedand were PEGylated at that site. A few of the hIFN polypeptides had anatural amino acid substitution at position 149 as well. A couple ofmutants were made using the NusA system described in Example 27 andcontain a methionine at the N-terminus (designated with “M-”). PEGASYS®was used as a control. TABLE 11 hIFN polypeptide % Relative ActivityT6pAF-30K PEG 25 M16 pAF-30K PEG 41 M-H34pAF-30K PEG 1220 M-G37pAF-30KPEG 455 N45 pAF-30K PEG 67 Q46 pAF-30K PEG 100 F64 pAF-30K PEG Noactivity observed E78 pAF-30K PEG 111 Y85 pAF-30K PEG No activityobserved E87 pAF-30K PEG 32 E96 pAF-30K PEG 20 Q101 pAF-30K PEG 80 T108pAF-30K PEG 571 M111 pAF-30K PEG 89 D114 pAF-30K PEG 114 A118 pAF-30KPEG 72 Q124 pAF-30K PEG 385 A145 pAF-30K PEG No activity observed R149pAF-30K PEG No activity observed N156 pAF-30K PEG 49 R149Y/E107 pAF-30KPEG No activity observed R149S/E107 pAF-30K PEG No activity observedR149E/E107 pAF-30K PEG No activity observed PEGASYS ® 100

TABLE 12 hIFN polypeptide % Relative Activity E107 pAF-30K PEG 640 E107pAF-40K PEG 62 PEGASYS ® 100

Example 31

This example details the measurement of hIFN activity and affinity ofhIFN polypeptides for the hIFN receptor. The hIFN polypeptides describedin this Example were generated using the NusA system in Example 27.Natural amino acid substitutions were made to the IFNα2a polypeptidesequence (SEQ ID NO: 2) based on a sequence comparison between limitinand IFNα2a. The “M-” indicates that methionine was the first amino acidin the polypeptide. The M-IFN “HV” or “HV” mutant was generated with thefollowing substitutions in hIFNα-2a (SEQ ID NO: 2): D77-D94 is replacedwith the mouse limitin sequence HERALDQLLSSLWRELQV. The M-IFN “CD” or“CD” mutant was generated with the following substitutions in hIFNα-2a(SEQ ID NO: 2): V105-D114 with GQSAPLP.

Biacore Studies (Receptor Binding Affinity)

The sequence for the IFNAR2 extracellular domain (consisting of 206amino acids ending with sequence LLPPGQ) was amplified from cloneMHS1011-61064 (OpenBiosystems, Huntsville, Ala.). This insert was clonedinto the pET20 expression vector (Novagen) downstream of the T7promoter. Protein expression was induced with 0.4 mM IPTG in BL21(DE3)cells (Novagen).

Since the expressed protein was insoluble, the inclusion bodies werepurified from lysed cells and solubilized in 6M GndCl. A 5 ml aliquot(50 mg amount) was reduced with 10 mM DTT for 45 minutes at 37° C. Thenthe mixture was injected into 200 ml of refolding buffer which consistedof 50 mM Tris pH 8, 20 mM NaCl, 0.5 M Arginine, 10% glycerol at 4° C.and incubated overnight with gentle stirring.

The refolding reaction was then concentrated to 25 ml using an Amiconstirring cell, and dialyzed overnight against 20 mM Tris, pH 8, 20 mMNaCl, 10% glycerol. Monomeric refolded IFNAR ECD was purified on HP QSepharose using the AKTA FPLC system (Amersham). Purified IFNAR2 ECD wasimmobilized on CM5 Biacore chip using a lysine-specific couplingprocedure recommended by the manufacturer. About 200 RUs of functionalprotein were immobilized. Various concentrations of IFN variants inHBS-EP buffer (Biacore) were injected at a flow rate of 50 mcl/minuteover the flowcell containing immobilized IFNAR2, and a control flowcellcontaining immobilized bovine serum albumin. Sensograms generated werefit to the 1:1 interaction model to calculate k_(on), k_(off) and K_(d)values using BiaEvaluation software (Biacore). Methionyl wild-typeinterferon (M-WT) was included as a control sample.

Table 13 shows the k_(on), k_(off), and K_(d) obtained with hIFNpolypeptides comprising one or more natural amino acid substitution.These mutants were made using the NusA system described in Example 27and contain a methionine at the N-terminus (designated with an “M-”).The sequences for the mutants were generated based on amino aciddifferences found between IFNα2a and limitin.

Table 14 shows the k_(on), k_(off), and K_(d) obtained with hIFNpolypeptides comprising a non-natural amino acid substitution(para-acetylphenylalanine) that was PEGylated with the linear 30K PEGshown in FIG. 19. These mutants were made using the NusA systemdescribed above and contain a methionine at the N-terminus (designatedwith an “M-”). TABLE 13 Binding parameters for IFNα2A:IFNAR2interaction, as determined by SPR IFNα2A k_(on), × 10⁻⁴ 1/M * s k_(off),1/s K_(d), nM M-WT 6.4 0.025 4 M-G10E 6 0.03 5 M-M16R 11 0.012 1.1M-T79R 9.2 0.026 2.8 M-K83Q 5.1 0.027 5.4 M-K83S 3.7 0.029 7.7 M-Y85L7.3 0.051 7 M-T86S 6.6 0.024 3.6 M-E87S 8 0.066 9 M-Q90R 9.9 0.021 2.1M-Q91E 5.5 0.041 7.5 M-D94V 7 0.017 2.4 M-E96K 7.5 0.025 3.3 M-R120K 5.50.026 4.6 M-K121T 6 0.027 4.5 M-Q124R/R125G 6.5 0.023 3.5 M-L128R 8.30.026 3.1 M-IFN “HV” 7.5 0.056 7.5 M-N93Q 4.9 0.027 5.5 M-IFN “CD” 6.50.022 3.3 M-M16R/Q90R/D94V 52 0.03 0.58 M-M16R/Q20R 21 0.022 1M-R149E >10 uM

TABLE 14 Binding parameters for IFNα2A:IFNAR2 interaction, as determinedby SPR IFNα2A k_(on), × 10⁻⁶ 1/M * s k_(off), 1/s K_(d), nM M-M16pAF-30K PEG 0.084 0.038 460 M-I24 pAF-30K PEG 0.112 0.016 145 M-F27pAF-30K PEG 0.09 0.0086 96 M-H34 pAF-30K PEG 0.18 0.019 105 M-G37pAF-30KPEG 0.197 0.047 240Anti-Viral Assays

To evaluate hIFN polypeptide anti-viral activity, 3×10⁴ human WISH cells(ATCC) were seeded in a 96 well/plate and were subsequently infectedwith 10,000 PFU of VSV per well. At the time of infection, differentamounts of hIFN polypeptide were added. 48 hours post-infection the CPEwas evaluated; 42-48 hours was the minimum time required to obtain 100%CPE. CPE was identified by staining the cells with 0.1 ml of 1 mg/ml3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT)followed by spectrophotometric reading at 570 nm with 690 nm as thereference wavelength. MTT measures metabolic reduction in mitochondria.

Table 15 shows the % relative anti-viral activity based on the IC₅₀values obtained with hIFN polypeptides that comprise one or more naturalamino substitutions. TABLE 15 hIFN polypeptide % Relative ActivityM-G10E 80 M-M16R 177 M-T79R 91 M-K83Q 72 M-K83S 106 M-Y85L 53 M-T86S 114M-E87S 90 M-Q90R 800 M-Q91E 38 M-N93Q 90 M-D94V 200 M-E96K 50 M-R120K105 M-K121T 52 M-L128R 70 M-R149E No activity observed M-Q124R/R125G 166M-M16R/Q20R 500 M-M16R/Q90R/D94V 0800 M-IFN “HV” 142 M-IFN “CD” 70 M-WT142 ROFERON ® 100

Example 32

This example details the measurement of hIFN activity and affinity ofhIFN polypeptides for the hIFN receptor. The hIFN polypeptides describedin this Example were generated using the following method.

Purification by HIC Column

500 ml LB Shake Flask Expression:

For each hIFN polypeptide, E. coli transformed with a construct encodingan orthogonal tRNA (J17 described in U.S. Patent Publication No.20030108885) and an orthogonal aminoacyl tRNA synthetase (E9 describedin U.S. patent application Ser. No. 11/292,903 entitled “Compositions ofAminoacyl-tRNA Synthetase and Uses Thereof”) with a mutated hIFNpolynucleotide were streaked onto a plate to obtain colonies. 2 mL LBcontaining antibiotic was inoculated with a single colony from thefreshly transformed plate. The culture was incubated under constantagitation (250 rpm) at 37° C. until the culture reached an OD600 ofapproximately 0.05-0.3 (approximately 5-6 hours). 500 mL LB (containingantibiotic and 4 mM pAF) was inoculated with all or part of the 2 mLculture, normalizing the inoculation to synchronize cultures. Theculture was incubated under constant agitation (250 rpm) at 37° C. untilculture reached an OD600 of approximately 1.0 (approximately 4-5 hours).The culture was then induced with 1 mM IPTG, and the culture wasincubated overnight at 37° C. The cells were harvested by centrifugationat 8000 rpm/12000 g; 15 minutes; 4° C. The cell paste was scraped outand stored in 40 mL Oak Ridge tubes. The cell pellets were then frozenat −80° C. until the inclusion body preparation was initiated.

Inclusion Body Preparation from 500 mL Shake Flask:

Buffer 1 was 50 mM NaAc pH 6.0; 100 mM NaCl; 1 mM EDTA; 1.0% TritonX-100. Buffer 2 was 50 mM NaAc pH 6.0; 100 mM NaCl; 1 mM EDTA. Table 16shows the procedure used for inclusion body preparation. TABLE 16 VolumeCentrifugation # Step Technique Buffer (mL) (g, minutes) 1 Lysis AvestinC3, 2 1 23 20000, 15 passes 2 Wash 1 Sonication, 30 1 25 20000, 15seconds 3 Wash 2 Sonication, 30 1 25 20000, 15 seconds 4 Rinse 1Sonication, 30 2 25 20000, 15 seconds 5 Rinse 2 Sonication, 30 2 2520000, 15 seconds

For the Lysis step, cells were resuspended in 23 mL chilled Buffer 1 byshaking for 1 hour at 4° C. The Avestin C3 was cleaned and rinsed with100 mL water and then with 30 mL Buffer 1. The cells were thenhomogenized with 2 passes in Avestin at 15,000-20,000 psi with coolingat 4° C. in the following manner: 1) The sample was added. 2) The samplewas processed once until Avestin reservoir was nearly empty; the samplewas collected in the original tube (also done for subsequent steps). 3)The collected sample was added back to the Avestin reservoir andre-homogenized. 4) The sample was washed out by homogenizing 12 mLBuffer 1; this was added to the sample. 5) The sample was transferred to40 mL Oak Ridge Centrifuge tube. 6) The sample was stored and 100 mLwater was run through the Avestin, adding it slowly while running toflush the system. 7) 30 mL Buffer 1 was then added to the reservoir andhomogenized to prepare for next sample. 8) The steps were then repeatedto process the next sample.

For the Wash and Rinse steps, a spatula was used to loosen the pelletoff the tube after pouring off supernatant from the last wash andpouring in fresh buffer. Sonication was performed at 75% power with anormal tip, not a microtip. The tip was rinsed with water and wiped witha Kimwipe between samples.

During the first 4 steps, the samples were spun in 40 mL Oak Ridge tubesused to store cell pellets. Rinse 2 was spun by splitting each sample to2 15 mL conical tubes.

Solubilization and Refolding:

Solubilization was done with 8M GndHCl pH between 5.5-8.5 using 50 mMNaAc or Tris. The inclusion bodies were solubilzed to a finalconcentration between 5-10 mg/mL. A final concentration of 10 mMβ-Mercaptoethanol was added, and the sample was incubated at roomtemperature for 30-60 minutes. The material was refolded or stored at−80° C. for long term storage.

The refolding was performed by diluting the solubilized material to afinal total protein concentration of 0.5 mg/mL in 4° C. Refold Buffer(50 mM Tris, pH 8.3; 0.5M Arginine). The refolding mixture was stored at4° C. for 2-4 days.

HIC Purification:

hIFN polypeptide refold samples were allowed to warm to roomtemperature. A final concentration of 1.5M NaCl was added to the sample,and the sample was incubated at room temperature for 30-60 minutes.Samples were filtered with a 0.22 μM PES filter and loaded onto a Butyl650M column (Tosoh) equilibrated in HIC Buffer A (20 mM sodiumphosphate, pH 7.0; 1.5M NaCl; 2M Urea). A linear gradient to 100% HICBuffer B (20 mM sodium phosphate, pH 7.0; 2M Urea) was run over 20column volumes.

SP HP Purification of HIC Pool:

The IFN polypeptide peak was collected from the Butyl column and dilutedto less than 30 m/S with water. The pH of samples were adjusted to 3.0with HCl and were loaded onto a SP HP column (GE Healthcare)equilibrated in SP Buffer A1 (50 mM NaAc, pH 3.0; 1 mM EDTA). After thesample was loaded, the column was washed with 2 column volumes of SPBuffer A followed by 4 column volumes of SP Buffer A2 (50 mM NaAc, pH5.0; 1 mM EDTA). A linear gradient was run from 100% SP Buffer A2 to 50%SP B Buffer (50 mM NaAc, pH 5.0; 0.5M NaCl; 10% ethylene glycol; 1 mMEDTA) over 15 column volumes.

PEGylation and Purification:

The hIFN polypeptide samples were pooled from the SP HP column andconcentrated to 1.0-2.0 mg/mL. 10% Acetic Acid was added to the sampleto drop the pH down to 4.0. Oxyamino-derivatized 30K PEG was added tohIFN polypeptide at a 1:12 molar ratio (30 mg/mg IFN/ml), and themixture was incubated at 28° C. for 48-72 hours. FIG. 19 shows the 30KPEG used in the conjugation. The sample was diluted ten fold with SPBuffer A (50 mM Na acetate, pH 5; 1 mM EDTA). To purify the PEGylatedhIFN, a SP HP column (GE Healthcare) was used with a gradient of 0-50%SP buffer B (50 mM Na acetate, pH 5, 0.5 M NaCl, 10% ethylene glycol; 1mM EDTA) over 15 column volumes. PEGylated IFN eluted around 100 mMsalt, followed by non-PEGylated monomer (baseline separation). Thefractions of PEGylated hIFN were pooled, dialyzed against the followingstorage buffer: 20 mM NaAcetate, pH 6, 0.005% Tween, 125 mM NaCl.Samples were concentrated to 1.0-2.0 mg/mL and stored at −80° C.

Biacore Studies (Receptor Binding Affinity)

The sequence for the IFNAR2 extracellular domain (consisting of 206amino acids ending with sequence LLPPGQ) was amplified from cloneMHS1011-61064 (OpenBiosystems, Huntsville, Ala.). This insert was clonedinto the pET20 expression vector (Novagen) downstream of the T7promoter. Protein expression was induced with 0.4 mM IPTG in BL21(DE3)cells (Novagen).

Since the expressed protein was insoluble, the inclusion bodies werepurified from lysed cells and solubilized in 6M GndCl. A 5 ml aliquot(50 mg amount) was reduced with 10 mM DTT for 45 minutes at 37° C. Thenthe mixture was injected into 200 ml of refolding buffer which consistedof 50 mM Tris pH 8, 20 mM NaCl, 0.5 M Arginine, 10% glycerol at 4° C.and incubated overnight with gentle stirring.

The refolding reaction was then concentrated to 25 ml using an Amiconstirring cell, and dialyzed overnight against 20 mM Tris, pH 8, 20 mMNaCl, 10% glycerol. Monomeric refolded IFNAR ECD was purified on HP QSepharose using the AKTA FPLC system (Amersham). Purified IFNAR2 ECD wasimmobilized on CM5 Biacore chip using a lysine-specific couplingprocedure recommended by the manufacturer. About 200 RUs of functionalprotein were immobilized. Various concentrations of IFN variants inHBS-EP buffer (Biacore) were injected at a flow rate of 50 mcl/minuteover the flowcell containing immobilized IFNAR2, and a control flowcellcontaining immobilized bovine serum albumin. Sensograms generated werefit to the 1:1 interaction model to calculate k_(on), k_(off) and K_(d)values using BiaEvaluation software (Biacore). Methionyl wild-typeinterferon (M-WT) and PEGASYS® were included as control samples.

Table 17 shows the k_(on), k_(off), and K_(d) obtained with hIFNpolypeptides comprising the non-naturally encoded amino acid pAF andwere PEGylated at the non-naturally encoded amino acid with the linear30K PEG as shown in FIG. 19. In addition, some of the hIFN polypeptidestested have a natural amino acid substitution. For example, T79R/N45pAcF −30K PEG was generated by substituting the non-natural amino acidpAF at position 45 of SEQ ID NO: 2 and the threonine amino acid atposition 79 was substituted with the naturally encoded amino acidarginine. TABLE 17 Binding parameters for IFNα2A:IFNAR2 interaction, asdetermined by SPR k_(on), × 10⁻⁴ K_(d), IFNα2A 1/M * s k_(off), 1/s nMM-WT 640 0.025 4 PEGASYS ® 6.9 0.027 390 N45 pAF-30K PEG 11 0.035 330T79R/N45 pAF-30K PEG 17 0.042 240 Y85L/N45 pAF-30K PEG 16 0.077 480E87S/N45 pAF-30K PEG 15 0.117 800 Q46 pAF-30K PEG 11 0.03 270 T79R/Q46pAF-30K PEG 15 0.032 210 Y85L/Q46 pAF-30K PEG 16.5 0.07 420 E87S/Q46pAF-30K PEG 15 0.093 630 E107 pAF-30K PEG 14.5 0.029 200 T79R/E107pAF-30K PEG 17 0.03 170 Y85L/E107 pAF-30K PEG 20 0.065 330 Y85S/E107pAF-30K PEG 19 0.057 300 E87S/E107 pAF-30K PEG 18 0.09 500 L80 pAF-30KPEG 6.7 0.034 500 Y89 pAF-30K PEG 14 0.029 200Anti-Viral Assays

To evaluate hIFN polypeptide anti-viral activity, 3×10⁴ human WISH cells(ATCC) were seeded in a 96 well/plate and were subsequently infectedwith 10,000 PFU of VSV per well. At the time of infection, differentamounts of hIFN polypeptide were added. 48 hours post-infection the CPEwas evaluated; 42-48 hours was the minimum time required to obtain 100%CPE. CPE was identified by staining the cells with 0.1 ml of 1 mg/ml3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT)followed by spectrophotometric reading at 570 nm with 690 nm as thereference wavelength. MTT measures metabolic reduction in mitochondria.

Table 18 shows the % relative anti-viral activity based on the IC₅₀values obtained with hIFN polypeptides that have a non-natural aminosubstitution at the site indicated and were PEGylated at that site. hIFNpolypeptides with a lower IC₅₀ value show higher % relative activity.Some of the PEGylated hIFN polypeptides in addition had a naturallyencoded amino acid substitution (T79R; Y85L; E87S; Y85S). TABLE 18 %Relative hIFN polypeptide Activity PEGASYS ® 100 N45pAF-30K PEG 300T79R/N45pAF-30K PEG 529 Y85L/N45pAF-30K PEG 150 E87S/N45pAF-30K PEG 139Q46pAF-30K PEG 395 T79R/Q46pAF-30K PEG 441 Y85L/Q46pAF-30K PEG 188E87S/Q46pAF-30K PEG 197 E107pAF-30K PEG 740 T79R/E107pAF-30K PEG 728Y85L/E107pAF-30K PEG 336 Y85S/E107pAF-30K PEG 176 E87S/E107pAF-30K PEG268 L80pAF-30K PEG 145 Y89pAF-30K PEG 176

Example 33 Measurement of Anti-Proliferative Activity

hIFN polypeptides described in the previous examples (Examples 30-32)were also assayed for anti-proliferative activity. A prominent effect ofIFNα's is their ability to inhibit cell growth, which is of majorimportance in determining anti-tumor action. The human lymphoblastoidDaudi cell line has proven to be extremely sensitive to IFNα's, and ithas been used to measure antiproliferative activity in many IFNα's andderived hybrid polypeptides (Meister et al., J Gen Virol. (1986) August;67 (Pt 8):1633-43). Use of this cell line has been facilitated by itsability to be grown in suspension cultures (Evinger and Pestka, (1981)Methods Enzymol. 79:362-368).

The human Daudi B cell line was purchased from ATCC (Manassas, Va.) andgrown in RPMI 1640 supplemented with 10% heat inactivated fetal bovineserum (Hyclone, Logan, Utah). The cell culture was maintained at 37° C.in a humidified atmosphere of 5% CO₂.

The Daudi cells were plated at a density of 1×10⁴ cells/well in aflat-bottom, 96-well plate. The cells were treated with increasingconcentration of IFNα2A in triplicates per dose concentration in a finalvolume of 200 ul. Following a 4-day incubation period at 37° C. with 5%CO₂, 20 ul of WST-1 solution Reagent (Roche cat#1644807) was added toeach well, and the culture was allowed to incubate for an additional 6hours. Absorbance was read at 440 nm using a Spectromax. IC₅₀s wereobtained from dose response curves plotted with OD 440 nm (average oftriplicates) against protein concentration with SigmaPlot.

Table 19 shows the results obtained with different hIFN molecules thatare nonPEGylated and PEGylated. “Cu” indicates polypeptides that weremade using the copper column as described in Example 30. “HIC” indicatespolypeptides that were made using the HIC method as described in Example32. TABLE 19 PEG hIFN polypeptide IC₅₀ % PEGASYS ® 30K PEG PEGASYS ®0.41 100%  40K PEG E107 pAF 0.84 206%  30K PEG E107 pAF Cu 0.04 10%  30KPEG T108 pAF 0.09 22%  30K PEG D114 pAF 0.27 66%  30K PEG A118 pAF 0.4098%  30K PEG M111 pAF 0.26 64%  30K PEG Q101 pAF 0.28 69%  30K PEG N156pAF 0.56 137%  30K PEG E96 pAF 1.27 311%  30K PEG Q46 pAF 0.14 34%  30KPEG E78 pAF 0.22 54%  30K PEG E87 pAF 0.86 211%  30K PEG N45 pAF 0.3483%  30K PEG M16 pAF 0.52 127%  30K PEG T6 pAF 0.79 193%  30K PEGR149Y/E107 pAF 3.84 940%  No PEG M-Q90R 0.01 3% No PEG M-D94V 0.01 3% NoPEG M-IFN “HV” 0.02 5% No PEG M-Q124R/R125G 0.01 3% No PEG M-K83S 0.025% No PEG M-WT 0.02 4% No PEG M-N93Q 0.01 3% No PEG M-T86S 0.01 3% NoPEG M-K121T 0.02 5% No PEG M-R120K 0.01 3% No PEG M-K83Q 0.03 7% No PEGM-M16R 0.01 2% No PEG M-E87S 0.02 4% No PEG ROFERON ® 0.01 3% No PEGM-IFN “CD” 0.01 3% No PEG M-G10E 0.01 3% No PEG M-T79R 0.01 4% No PEGM-Y85L 0.03 7% No PEG M-E96K 0.13 32%  No PEG M-R149E* dead dead No PEGSigma WT IFN 0.01 2% 30K PEG N45 pAF Cu 0.22 55%  30K PEG N45 pAF HIC0.07 17%  30K PEG Q46 pAF Cu 0.12 30%  30K PEG Q46 pAF HIC 0.12 28%  30KPEG L80 pAF 0.08 19%  30K PEG Y89 pAF 0.11 27%  30K PEG E107pAF HIC 0.037% 30K PEG T79R/N45 pAF 0.04 10%  30K PEG Y85L/E107 pAF 0.06 14%  30KPEG Y85S/E107 pAF 0.12 30% 

Example 34 Colony Formation Assays

Colony formation assays such as those described by Giron-Michel, J. inLeukemia 2002 16:1135-1142 may be used to evaluate proliferation ofprogenitor cells by hIFN polypeptides of the invention. Cord blood maybe used in colony formation assays.

Evaluation of the Toxicity of hIFN Polypeptides on Human Myeloid andErythroid Progenitors

Using a methylcelluose-based in vitro colony forming assay, thehematopoietic toxicity of twenty one nonPEGylated compounds (twenty hIFNpolypeptides with Roferon® as control) and eighteen PEGylated compounds(seventeen polypeptides PEGylated with a 30K PEG with PEGASYS® as acontrol) were tested. The nonPEGylated hIFN polypeptides were made bythe NusA method shown in Example 27 and were characterized as shown inExample 31. PEGylated hIFN polypeptides were generated and characterizedas shown in Examples 30 and 31.

Cells: Normal human bone marrow light density cells (Poietics Inc.,Maryland) were stored at −152° C. until required for the assay. On theday of the experiment, the cells were thawed rapidly at 37° C., and thecontents of the vial were diluted in 10 mls of Iscove's mediumcontaining 2% fetal bovine serum and washed by centrifugation. Thesupernatant was discarded, and the cell pellet resuspended in a knownvolume of Iscove's medium containing 2% FBS. A cell count (3% glacialacetic acid) and viability assessment by Trypan Blue exclusion wasperformed.

Test samples: Compounds were in a buffer of 20 mM NaAc, 125 mM NaCl,0.005% Tween 80, pH 6.0. Each compound was tested in ten testconcentrations in a series of five fold dilutions. Triplicates wereperformed at each dose concentration. Dilutions of each compound wereprepared with the same buffer to generate the required dilutions. Whenadded to methylcellulose, the buffer was at a final concentration of6.7% and compound concentrations ranged from 5—0.00000256 ug/ml fornonPEGylated hIFN test polypeptides and 26—0.0000133 ug/ml for 30KPEGylated hIFN test polypeptides. In instances where the initial 30KPEGylated hIFN test polypeptides concentrations are low, a concentrationrange of 14—0.000007168 ug/ml was adopted instead.

Method: Clonogenic progenitors of the erythroid (CFU-E and BFU-E),granulocyte-monocyte (CFU-GM) and multipotential (CFU-GEMM) lineageswere assessed in methylcellulose-based medium (MethoCult™ 4434)containing saturating concentrations of the cytokines SCF (Stem CellFactor; 50 ng/mL), GM-CSF (Granulocyte Macrophage Colony StimulatingFactor; 10 ng/mL), IL-3 (Interleukin 3; 10 ng/mL), and EPO(Erythropoietin; 3 U/mL). CFU-E is a small erythroid colony derived fromthe most mature erythroid colony forming cells. It contains one to twoclusters with a total number of 8-200 erythroblasts. BFU-E is a largererythroid colony derived from a more primitive cell. It contains greaterthan 200 erythroblasts. CFU-GM is a colony that is derived from a colonyforming cell capable of producing colonies with forty or moregranulocyte-monocyte and/or macrophage cells. CFU-GEMM is a colony thatcontains cells from more than one lineage. It is derived from the mostprimitive colony forming cell and contains erythroid cells as well astwenty or more granulocytes, macrophages, and megakaryocytes. At eachassay set up, a standard control containing MethoCult™ 4434, cells andmedia and a buffer control containing MethoCult™ 4434, cells andequivalent amount of buffer but no compound were included to establish acondition in which no toxicity should be observed.

Clonogenic progenitors of the erythroid (CFU-E and BFU-E),granulocyte-monocyte/myeloid (CFU-GM) and multipotential (CFU-GEMM)lineages were set up in the methylcellulose-based media described. Thecompounds were added to the MethoCult™ to give final concentrations asdescribed above. Vehicle control cultures containing no compound butequivalent concentrations of vehicle buffer as well as standard controlscontaining no compounds or vehicle buffer but media alone were alsoinitiated. All cultures were set up in triplicate at 1×10⁴ cells perculture. Following 14 days in culture, the colonies were assessed andscored. The colonies were divided into the following categories, basedon size and morphology: CFU-E, BFU-E, CFU-GM, and CFU-GEMM.

Results and analysis: Triplicate cultures for CFU-E, BFU-E, CFU-GM, andCFU-GEMM were enumerated. In addition, the distribution of colony typesas well as general colony and cellular morphology were analyzed. In alltest sets, no difference between the numbers of colonies generated instandard cultures compared to the buffer controls were observed.However, in the presence of each nonPEGylated or PEGylated hIFNpolypeptides, colony numbers as well as colony size of both the myeloidand erythroid lineage were perturbed when compared to standard andbuffer assay controls. The morphology of the resulting erythroid andmyeloid colonies was however not affected. A dose dependent toxic effecton both myeloid and erythroid progenitors were seen for all nonPEGylatedand PEGylated hIFN tested including compound controls Roferon® andPEGASYS®.

Further analysis of test compounds were performed to normalize fordifferences in anti-viral activity observed between compounds. Massconcentrations of hIFN polypeptides used in the colony formation assaywere divided by the anti-viral IC₅₀ values measured in the VSVreplication assay. The myeloid and erythroid colony counts were thenplotted on Y-axis against the normalized protein concentration on a logx-axis. Relative toxicity of each test polypeptides compared to theappropriate compound control was determined by the factor rho where rhorepresents the shift in x-axis between two curves. When rho>1, testcompound is more potent and hence more toxic than control. When rho>1,the fold improvement was represented as a negative number that has thevalue of rho (−rho) (See Tables 22 and 23). When rho<1, test compound isless toxic than compound control. When rho<1, the fold improvement wasrepresented as a positive number that has the value of 1/rho (1/rho)(See Tables 20 and 21). For nonPEGylated hIFN polypeptides, Roferon® wasused as the compound control. For PEGylated hIFN polypeptides, PEGASYS®was used as the compound control. TABLE 20 Fold improvement in nonPEGCandidates toxicity M-G10E −1.69 M-M16R −1.97 M-T79R 2.33 M-K83Q −2.13M-K83S 1.71 M-Y85L 6.63 M-T86S 2.74 M-E87S 4.50 M-Q90R −5.03 M-Q91E 1.81M-N93Q −4.31 M-D94V −36.79 M-E96K −1.76 M-R120K −14.11 M-K121T −2.13M-Q124R/R125G −25.33 M-L128R −1.79 M-IFN “CD” −2.60 M-IFN “HV” −101.85

TABLE 21 Fold improvement in PEG Candidates toxicity T6pAF-30K PEG 1.56M16pAF-30K PEG 3.13 M-H34pAF 30K PEG −3.19 M-G37pAF 30K PEG 2.78 N45pAF30K PEG 5.26 Q46pAF 30K PEG 4.76 E78pAF 30K PEG 1.64 E87pAF 30K PEG 1.92E96pAF 30k PEG −11.06 Q101pAF 30K PEG −2.76 E107pAF 30K PEG 2.50 E107pAF40K PEG 1.67 T108pAF 30K PEG 2.38 M111pAF 30K PEG −1.24 D114pAF 30K PEG−1.83 A118pAF 30K PEG −2.89 N156pAF 30K PEG −2.77

Example 34 Comparison of hIFN Polypeptides

For the VSV assay, more active molecules have a lower IC₅₀ value. If theVSV IC₅₀ of the test compound was less than the VSV IC₅₀ of M-WT(wild-type interferon with a methionine at the N-terminus), then thefold improvement was expressed as VSV IC₅₀ M-WT/VSV IC₅₀ test compound.If the VSV IC₅₀ of the test compound was greater than the VSV IC₅₀ ofM-WT, then the fold improvement was expressed as −(VSV IC₅₀ testcompound/VSV IC₅₀ M-WT). Table 22 shows the fold improvement data for aset of hIFN polypeptides described also in Example 33. TABLE 22 Foldimprovement in VSV anti-viral nonPEG Candidates activity M-G10E −1.79M-M16R 1.24 M-T79R −1.55 M-K83Q −1.96 M-K83S −1.34 M-Y85L −2.68 M-T86S−1.25 M-E87S −1.61 M-Q90R 5.60 M-Q91E −3.75 M-N93Q −1.61 M-D94V 1.40M-E96K −3.00 M-R120K −1.36 M-K121T −2.71 M-Q124R/R125G 1.17 M-L128R−2.00 M-IFN “GD” −2.14 M-IFN “HV” 1.00

For the VSV assay, more active molecules have a lower IC₅₀ value. If theVSV IC₅₀ of the test compound was less than the VSV IC₅₀ of PEGASYS®,then the fold improvement was expressed as VSV IC₅₀ PEGASYS®/VSV IC₅₀test compound. If the VSV IC₅₀ of the test compound was greater than theVSV IC₅₀ of PEGASYS®, then the fold improvement was expressed as −(VSVIC₅₀ test compound/VSV IC₅₀ PEGASYS®). Table 23 shows the foldimprovement data for one set of PEGylated hIFN polypeptides describedalso in Example 33. TABLE 23 Fold improvement in VSV anti-viral PEGCandidates activity T6pAF-30K PEG −4.00 M16pAF-30K PEG −2.43 M-H34pAF30K PEG 12.20 M-G37pAF 30K PEG 4.55 N45pAF 30K PEG −1.50 Q46pAF 30K PEG1.00 E78pAF 30K PEG 1.11 E87pAF 30K PEG −3.13 E96pAF 30K PEG −5.00Q101pAF 30K PEG −1.25 E107pAF 30K PEG 4.95 E107pAF 40K PEG −1.73 T108pAF30K PEG 5.71 M111pAF 30K PEG −1.13 D114pAF 30K PEG 1.14 A118pAF 30K PEG−1.38 N156pAF 30K PEG −2.04

Table 24 shows a list of candidates with improved toxicity and theirassociated fold improvement in anti-viral activity. TABLE 24 Foldimprovement Fold anti-viral improvement hIFN polypeptide activity inToxicity M-T79R −1.55 2.33 M-K83S −1.34 1.71 M-Y85L −2.68 6.63 M-T86S−1.25 2.74 M-E87S −1.61 4.50 M-Q91E −3.75 1.81 T6pAF 30K PEG −4.00 1.56M16pAF 30K PEG −2.43 3.13 M-G37pAF 30K PEG 4.55 2.78 N45pAF 30K PEG−1.50 5.26 Q46pAF 30K PEG 1.00 4.76 E78pAF 30K PEG 1.11 1.64 E87pAF 30KPEG −3.13 1.92 E107pAF 30K PEG 4.95 2.50 T108pAF 30K PEG 5.71 2.38

Example 35 Human Clinical Trial of the Safety and/or Efficacy ofPEGylated hIFN Comprising a Non-Naturally Encoded Amino Acid

A Phase 0 study is performed to investigate microdosing of hIFNpolypeptides of the invention. This study involves a small number ofsubjects. These studies involve measuring changes in molecular markerswith administration of a fraction of the therapeutic dose. These samemarkers correlate with a therapeutic response when the drug isadministered to an affected individual. The drug-response informationobtained is useful for Phase I and Phase II trial design.

Objective To compare the safety and pharmacokinetics of subcutaneouslyadministered PEGylated recombinant human hIFN comprising a non-naturallyencoded amino acid with the commercially available hIFN products RoferonA® or Intron A®.

Patients Eighteen healthy volunteers ranging between 20-40 years of ageand weighing between 60-90 kg are enrolled in the study. The subjectswill have no clinically significant abnormal laboratory values forhematology or serum chemistry, and a negative urine toxicology screen,HIV screen, and hepatitis B surface antigen. They should not have anyevidence of the following: hypertension; a history of any primaryhematologic disease; history of significant hepatic, renal,cardiovascular, gastrointestinal, genitourinary, metabolic, neurologicdisease; a history of anemia or seizure disorder; a known sensitivity tobacterial or mammalian-derived products, PEG, or human serum albumin;habitual and heavy consumer to beverages containing caffeine;participation in any other clinical trial or had blood transfused ordonated within 30 days of study entry; had exposure to hIFN within threemonths of study entry; had an illness within seven days of study entry;and have significant abnormalities on the pre-study physical examinationor the clinical laboratory evaluations within 14 days of study entry.All subjects are evaluable for safety and all blood collections forpharmacokinetic analysis are collected as scheduled. All studies areperformed with institutional ethics committee approval and patientconsent.

Study Design This will be a Phase I, single-center, open-label,randomized, two-period crossover study in healthy male volunteers.Eighteen subjects are randomly assigned to one of two treatment sequencegroups (nine subjects/group). IFN is administered over two separatedosing periods as a bolus s.c. injection in the upper thigh usingequivalent doses of the PEGylated hIFN comprising a non-naturallyencoded amino acid and the commercially available product chosen. Thedose and frequency of administration of the commercially availableproduct is as instructed in the package label. Additional dosing, dosingfrequency, or other parameter as desired, using the commerciallyavailable products may be added to the study by including additionalgroups of subjects. Each dosing period is separated by a 14-day washoutperiod. Subjects are confined to the study center at least 12 hoursprior to and 72 hours following dosing for each of the two dosingperiods, but not between dosing periods. Additional groups of subjectsmay be added if there are to be additional dosing, frequency, or otherparameter, to be tested for the PEGylated hIFN as well. Multipleformulations of IFN that are approved for human use may be used in thisstudy. Roferon A® and/or Intron A® are commercially available IFNproducts approved for human use. The experimental formulation of hIFN isthe PEGylated hIFN comprising a non-naturally encoded amino acid.

Blood Sampling Serial blood is drawn by direct vein puncture before andafter administration of hIFN. Venous blood samples (5 mL) fordetermination of serum IFN concentrations are obtained at about 30, 20,and 10 minutes prior to dosing (3 baseline samples) and at approximatelythe following times after dosing: 30 minutes and at 1, 2, 5, 8, 12, 15,18, 24, 30, 36, 48, 60 and 72 hours. Each serum sample is divided intotwo aliquots. All serum samples are stored at −20° C. Serum samples areshipped on dry ice. Fasting clinical laboratory tests (hematology, serumchemistry, and urinalysis) are performed immediately prior to theinitial dose on day 1, the morning of day 4, immediately prior to dosingon day 16, and the morning of day 19.

Bioanalytical Methods An ELISA kit procedure (BioSource International(Camarillo, Calif.)), is used for the determination of serum IFNconcentrations.

Safety Determinations Vital signs are recorded immediately prior to eachdosing (Days 1 and 16), and at 6, 24, 48, and 72 hours after eachdosing. Safety determinations are based on the incidence and type ofadverse events and the changes in clinical laboratory tests frombaseline. In addition, changes from pre-study in vital signmeasurements, including blood pressure, and physical examination resultsare evaluated.

Data Analysis Post-dose serum concentration values are corrected forpre-dose baseline IFN concentrations by subtracting from each of thepost-dose values the mean baseline IFN concentration determined fromaveraging the IFN levels from the three samples collected at 30, 20, and10 minutes before dosing. Pre-dose serum IFN concentrations are notincluded in the calculation of the mean value if they are below thequantification level of the assay. Pharmacokinetic parameters aredetermined from serum concentration data corrected for baseline IFNconcentrations. Pharmacokinetic parameters are calculated by modelindependent methods on a Digital Equipment Corporation VAX 8600 computersystem using the latest version of the BIOAVL software. The followingpharmacokinetics parameters are determined: peak serum concentration(C_(max)); time to peak serum concentration (t_(max)); area under theconcentration-time curve (AUC) from time zero to the last blood samplingtime (AUC₀₋₇₂) calculated with the use of the linear trapezoidal rule;and terminal elimination half-life (t_(1/2)), computed from theelimination rate constant. The elimination rate constant is estimated bylinear regression of consecutive data points in the terminal linearregion of the log-linear concentration-time plot. The mean, standarddeviation (SD), and coefficient of variation (CV) of the pharmacokineticparameters are calculated for each treatment. The ratio of the parametermeans (preserved formulation/non-preserved formulation) is calculated.

Safety Results The incidence of adverse events is equally distributedacross the treatment groups. There are no clinically significant changesfrom baseline or pre-study clinical laboratory tests or blood pressures,and no notable changes from pre-study in physical examination resultsand vital sign measurements. The safety profiles for the two treatmentgroups should appear similar.

Pharmacokinetic Results Mean serum IFN concentration-time profiles(uncorrected for baseline IFN levels) in all 18 subjects after receivinga single dose of commercially available hIFN (e.g. Roferon A® or IntronA®) are compared to the PEGylated hIFN comprising a non-naturallyencoded amino acid at each time point measured. All subjects should havepre-dose baseline IFN concentrations within the normal physiologicrange. Pharmacokinetic parameters are determined from serum datacorrected for pre-dose mean baseline IFN concentrations and the C_(max)and t_(max) are determined. The mean t_(max) for hIFN (e.g. Roferon®) issignificantly shorter than the t_(max) for the PEGylated hIFN comprisingthe non-naturally encoded amino acid. Terminal half-life values aresignificantly shorter for hIFN (e.g. Intron A®) compared with theterminal half-life for the PEGylated hIFN comprising a non-naturallyencoded amino acid.

Although the present study is conducted in healthy male subjects,similar absorption characteristics and safety profiles would beanticipated in other patient populations; such as male or femalepatients with cancer or chronic renal failure, pediatric renal failurepatients, patients in autologous predeposit programs, or patientsscheduled for elective surgery. Alternatively, the Phase I study isperformed in HCV subjects to evaluate PEGylated hIFN polypeptide.

In conclusion, subcutaneously administered single doses of PEGylatedhIFN comprising non-naturally encoded amino acid will be safe and welltolerated by healthy male subjects or HCV subjects. Based on acomparative incidence of adverse events, clinical laboratory values,vital signs, and physical examination results, the safety profiles ofhIFN (e.g. Roferon A®) and PEGylated hIFN comprising non-naturallyencoded amino acid will be equivalent. The PEGylated hIFN comprisingnon-naturally encoded amino acid potentially provides large clinicalutility to patients and health care providers.

Example 36

This example details methods used to generate hIFN polypeptidescomprising a non-naturally encoded amino acid. Purified refolded hIFNpolypeptides were conjugated to poly(ethylene glycol) according to themethods described below. Modifications to the methods described belowand alternate methods to refold polypeptides such as hIFN polypeptidesare known to those of ordinary skill in the art. Modifications to themethods described include, but are not limited to, the addition,substitution, or subtraction of one or more steps mentioned below.Potential methods that may be used or one or more modifications to themethods described include, but are not limited to, washing inclusionbodies with reagents that reduce the amount of contaminant proteins thatpotentially interact with the hIFN polypeptide such reagents include butare not limited to urea and deoxycholic acid; adding a reagent to therefolding reaction such reagents include but are not limited to adenaturant, guanidine HCl, urea, cysteine, and catalytic amounts of areducing agent. Columns other than those described may be used in toisolate or purify hIFN polypeptides comprising a non-naturally encodedamino acid. Such columns include but are not limited to a hydrophobicinteraction chromatography (HIC) column such as a butyl, octyl, orphenyl HIC column. Alternate conditions and solutions may be used forisolation or purification of hIFN polypeptides, including but notlimited to the use of alternate salts such as ammonium sulfate, sodiumcitrate, and other salts; the adjustment of buffer components includingbut not limited to, the adjustment of salt concentrations to facilitatebinding to a column used in purification/isolation; alterations in pH;the replacement of one or more reagents; and the inclusion of one ormore reagents, including but not limited to, arginine.

Alternate methods include but are not limited to, solubilization of cellpellets with guanidine and other methods known to those of ordinaryskill in the art. Different strains of bacteria may also be used in theexpression of hIFN polypeptides.

Purification of IFN Inclusion Bodies

For each hIFN polypeptide, E. coli strains BL21(A1), BL21(DE3) orW3110(B2) transformed with a construct encoding an orthogonal tRNA (J17described in U.S. Patent Publication No. 20030108885) and an orthogonalaminoacyl tRNA synthetase (E9 described in U.S. patent application Ser.No. 11/292,903 entitled “Compositions of Aminoacyl-tRNA Synthetase andUses Thereof”) with a mutated hIFN polynucleotide. The bacteria wereplated onto LB Agar with ampicillin plates and incubated overnight at37° C. Colonies were selected and grown in culture to make glycerolstocks.

5 ul aliquots of glycerol stock were used to make a 5 ml starter culturein the morning. The starter culture was added to LB media containingpara-acetylphenylalanine (pAF) in the morning (1×LB; 4 mM pAF; 1×AMP),and the cells were induced in the evening or when the culture had an ODof greater than 1.0. The culture was grown/induced at 37° C. degreeswith shaking at 250 rpm overnight.

The next morning the OD was measured of the cultures, and the cells werepelleted. The OD measurements of the cultures were between about 3 andabout 8. The cell pellet was either frozen at −80° C. or the inclusionbodies were prepared from the pellet immediately. An aliquot wasanalyzed by SDS-PAGE with a Coomassie stain to check expression and thelength of product produced. The bacterial pellet was resuspended in 35ml of IB1 buffer (50 mM NaAc, pH 6.0; 100 mM NaCl; 1 mM EDTA; 0.1%Triton X). Then the sample was sonicated six times to resuspend and lysethe cells. 30 second bursts of sonication were followed by 1 minuteincubations on ice. The bacterial lysate was centrifuged in a 50 mlOakridge tube at 13,000 rpm for 20 minutes at 4° C., and the supernatantwas discarded. The pellet was washed four times with IB1 buffer usingsonication to resuspend. The inclusion bodies were spun down, and thesupernatant was discarded. The pellet was washed two times with IB2buffer (50 mM NaAc, pH 6.0; 100 mM NaCl; 1 mM EDTA), and the supernatantwas discarded. The pellet was resuspended in 5-15 ml of 8M Guanidine HCl(8 M GnHCl; 50 mM NaAc pH 6.0) and were resuspended using a douncer. Theconcentration of the sample was determined using a 100× dilution, andthe OD was measured at 280 nm. The IFN extinction coefficient is 22800or 1.17. Sample concentrations were normalized to 5.0 mg/ml protein. Thepurity of the inclusion bodies was determined by SDS PAGE gel with aCoomasie stain. Twenty-seven hIFN polypeptides with apara-acetylphenylalanine substitution were suppressed, and inclusionbodies for twenty-seven mutants were isolated. Each of the twenty-sevenmutants generated had one non-naturally encoded amino acid substitutionin which para-acetylphenylalanine replaced a naturally encoded aminoacid in hIFN (SEQ ID NO: 2). The 27 hIFN polypeptides generated had asubstitution at one of the following positions: 23, 24, 25, 26, 27, 28,30, 31, 32, 33, 128, 129, 131, 132, 133, 134, 135, 136, 137, 158, 159,160, 161, 162, 163, 164, and 165.

Refolding hIFN Using Purified Inclusion Bodies

100 mg of 5.0 mg/ml hIFN was added to 400 ml Refolding Buffer (50 mMNaAc pH 6.0; 0.1% Tween 20) while spinning slowly at 4° C. Refolding wasperformed for 24 to 48 hours. The refolding mixture was then brought toroom temperature. Then 50 uM CuCl was added, and the sample was mixed.The sample was incubated for 15 minutes at room temperature.

Protein Purification and PEGylation

Any precipitate that formed during refolding was spun down or filtered,and the correctly folded hIFN polypeptide was purified using a copperchelate column. Copper Column Buffer A was 50 mM NaAc, pH 5.0; 0.15 MNaCl; 0.1% Tween 20. Copper Column Buffer B was 100 mM NaAc. pH 3.5;0.15 M NaCl; 0.1% Tween 20.

An aliquot was checked at this point by SDS-PAGE with a Commassie stain.Pooled fractions from the copper column were loaded onto a 5 ml SP HPcation exchange column. The 5 ml SP HP Buffer A was 50 mM NaAc pH 6.0; 1mM EDTA. The 5 ml SP HP buffer B was 50 mM NaAc pH 6.0; 1 mM EDTA; 500mM NaCl; 10% Ethylene glycol. The amount of total protein was measuredat this point using by measuring the OD at 280 nm, and SDS-PAGE with aCommassie stain was also performed.

Fractions from the copper chelate column were pooled and wereconcentrated to greater than 1.0 mg/ml and 10% acetic acid was added toeach sample to bring the pH to 4.0. The linear 30K PEG shown in FIG. 19was added at a 12:1 molar ratio. The sample was incubated at 28° C. for24 to 48 hours. The sample was then diluted 10-fold with Milli-Q water,and the PEGylated hIFN polypeptide was purified over a 5 ml SP HPcolumn. SDS-PAGE was performed with a Commassie stain to analyze thesample and determine the level of higher molecular weight species andunPEGylated contaminants. Fractions from the SP column were collected,and then the pool was dialyzed against Storage Buffer (25 mM NaAc, pH6.0; 120 mM NaCl; 0.005% Tween 80). An aliquot of the final product wasthen analyzed by SDS-PAGE. The final product was concentrated to 180ug/ml, and aliquots were stored at −80° C.

Example 37 VSV Anti-Viral Assay

Antiviral activity of hIFN polypeptides may be measured by a variety ofassays. The antiviral activity of PEGylated hIFN polypeptides wasdetermined using the Vesicular Stomatitis Virus (VSV). The culture mediafor this assay was DMEM, 10% FBS, 1% Penicillin/Streptomycin, 5 mlHEPES. Additional reagents included RPMI without FBS and Phenol Red. Theconcentration of MTT stock used was 5 mg/mL in PBS. This stock solutionwas stored for only two weeks at 4° C.

Human WISH cells were seeded at 30,000 cells/well in 50 μL of culturemedia. The following day, 2× serial dilutions were performed of the hIFNpolypeptides in culture media. PEGASYS® was used as a control in theseexperiments. In triplicate, 100 μL of the diluted hIFN polypeptides wasadded to the WISH cells for a final well volume of 150 μL. The cells andhIFN polypeptides were incubated for 6 hours at 37° C. in a CO₂incubator. After this six hour incubation, 10,000 PFU of VSV was addedper well, in a volume of 50 μL of media; 20 μL of VSV was diluted in 5ml of media per 96 well/plate. The media was removed by aspirationforty-five hours post-infection. The plates were gently tapped on papertowels to remove residual media.

50 μL of 1 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) prepared in RPMI without Phenol Red andFBS was added from a 5 mg/mL MTT stock solution. 1 mg/mL MTT was usuallyprepared fresh for every assay. Next, the plates were incubated forthree hours at 37° C. in the CO₂ incubator. The MTT was carefullyremoved by aspiration. 50 μL of isopropanol was added per well. Theplates were placed on a plate-shaker for 30 to 40 seconds to completethe formation of MTT. The plates were read at 560 nm with 690 nm as areference wavelength. The data was plotted and the IC₅₀ calculated usingthe Sigma-Plot program.

Table 25 summarizes the anti-viral activity of 20 hIFN polypeptides thatwere conjugated to 30K PEG via a para-acetylphenylalanine substitutionin the polypeptide. The second column lists the averaged IC₅₀ values foreach compound across multiple anti-viral activity experiments. The thirdcolumn is the % Efficacy relative to the averaged IC₅₀ values found inthe second column.

The fourth column is the Average % Efficacies. For this data, the %Efficacy was established by comparing IC₅₀s to the IC₅₀ of PEGASYS® inindividual experiments. Then the % Efficacies from all experiments wereaveraged. TABLE 25 AVE % Efficacy hIFN polypeptide AVE IC50 % Efficacy(individual expts.) PEGASYS ® 7.26 100% 100%  K23 pAF-30K PEG 7.18 101%95% I24 pAF-30K PEG 2.78 261% 261%  S25 pAF-30K PEG 27.51   26% 22% L26pAF-30K PEG No Activity No Activity No Activity observed observed to 50ng/mL observed to 50 ng/mL to 50 ng/mL F27 pAF-30K PEG 2.44 298% 277% S28 pAF-30K PEG not tested not tested not tested L30 pAF-30K PEG NoActivity No Activity No Activity observed observed to 50 ng/mL observedto 50 ng/mL to 50 ng/mL K31 pAF-30K PEG 4.58 159% 170%  D32 pAF-30K PEGnot tested not tested not tested R33 pAF-30K PEG No Activity No ActivityNo Activity observed observed to 50 ng/mL observed to 50 ng/mL to 50ng/mL L128 pAF-30K PEG 3.45 211% 200%  Y129 pAF-30K not tested nottested not tested PEG K131 pAF-30K 1.49 487% 536%  PEG E132 pAF-30K PEGnot tested not tested not tested K133 pAF-30K not tested not tested nottested PEG K134 pAF-30K 1.46 496% 560%  PEG Y135 pAF-30K 10.25   71% 76%PEG S136 pAF-30K PEG not tested not tested not tested P137 pAF-30K PEGnot tested not tested not tested Q158 pAF-30K 7.02 103% 107% PEG E159pAF-30K PEG 15.69   46% 39% S160 pAF-30K PEG 17.95   40% 35% L161pAF-30K PEG 13.99   52% 45% R162 pAF-30K 22.09   33% 27% PEG S163pAF-30K PEG 11.73   62% 55% K164 pAF-30K 9.96  73% 69% PEG E165 pAF-30KPEG 7.93  92% 81%

Example 38

This example details the measurement of hIFN activity and affinity ofhIFN polypeptides for the hIFN receptor.

Biacore Studies (Receptor Binding Affinity)

The sequence for the IFNAR2 extracellular domain (consisting of 206amino acids ending with sequence LLPPGQ) was amplified from cloneMHS1011-61064 (OpenBiosystems, Huntsville, Ala.). This insert was clonedinto the pET20 expression vector (Novagen) downstream of the T7promoter. Protein expression was induced with 0.4 mM IPTG in BL21 (DE3)cells (Novagen).

Since the expressed protein was insoluble, the inclusion bodies werepurified from lysed cells and solubilized in 6M GndCl. A 5 ml aliquot(50 mg amount) was reduced with 10 mM DTT for 45 minutes at 37° C. Thenthe mixture was injected into 200 ml of refolding buffer which consistedof 50 mM Tris pH 8, 20 mM NaCl, 0.5 M Arginine, 10% glycerol at 4° C.and incubated overnight with gentle stirring.

The refolding reaction was then concentrated to 25 ml using an Amiconstirring cell, and dialyzed overnight against 20 mM Tris, pH 8, 20 mMNaCl, 10% glycerol. Monomeric refolded IFNAR ECD was purified on HP QSepharose using the AKTA FPLC system (Amersham). Purified IFNAR2 ECD wasimmobilized on CM5 Biacore chip using a lysine-specific couplingprocedure recommended by the manufacturer. About 200 RUs of functionalprotein were immobilized. Various concentrations of IFN variants inHBS-EP buffer (Biacore) were injected at a flow rate of 50 mcl/minuteover the flowcell containing immobilized IFNAR2, and a control flowcellcontaining immobilized bovine serum albumin. Sensograms generated werefit to the 1:1 interaction model to calculate k_(on), k_(off) and K_(d)values using BiaEvaluation software (Biacore). PEGASYS® was included asa control sample. Table 26 shows the average k_(on), k_(off) and K_(d)obtained with the hIFN polypeptides characterized in Example 37. Thestandard deviations (SD) calculated for each value are also shown. TABLE26 hIFN k_(off) polypeptide k_(on) Average k_(on) SD Average k_(off) SDK_(d) Average K_(d) SD K31 pAF- 7.6 0.424264069 0.07 0.00 87070.71067812 30K PEG S163 pAF- 3.85 0.353553391 0.04 0.00 105070.71067812 30K PEG K164 pAF- 5.35 0.353553391 0.04 0.00 660 56.5685424930K PEG L26 pAF- No Binding No Binding No No No Binding No Binding 30KPEG observed observed Binding Binding observed observed observedobserved R33 pAF- No Binding No Binding No No No Binding No Binding 30KPEG observed observed Binding Binding observed observed observedobserved Q158 pAF- 6 0.141421356 0.03 0.00 440 21.21320344 30K PEG E159pAF- 2.9 0.141421356 0.03 0.00 1085 162.6345597 30K PEG Y135 pAF- 5.950.494974747 0.04 0.00 712.5 10.60660172 30K PEG L30 pAF- No Binding NoBinding No No No Binding No Binding 30K PEG observed observed BindingBinding observed observed observed observed K134 pAF- 7.4 0.4242640690.04 0.00 495 35.35533906 30K PEG L161 pAF- 4.95 0.212132034 0.05 0.00990 14.14213562 30K PEG K131 pAF- 9.9 0.141421356 0.03 0.00 33014.14213562 30K PEG I24 pAF-30K 4.95 0.070710678 0.02 0.00 39014.14213562 PEG S160 pAF- 4.825 0.106066017 0.05 0.00 1100 0 30K PEGE165 pAF- 5.35 0.070710678 0.03 0.00 577.5 3.535533906 30K PEG R162 pAF-4.65 0.777817459 0.06 0.00 1375 176.7766953 30K PEG PEGASYS ®7.491666667 0.537044381 0.03 0.00 368.3333333 31.88521078 K23 pAF- 5.455.727564928 0.13 0.00 1180 311.1269837 30K PEG S25 pAF- 7.95 2.8991378030.13 0.02 1700 848.5281374 30K PEG F27 pAF- 4.175 0.247487373 0.01 0.00220 14.14213562 30K PEG L128 pAF- 8.65 1.060660172 0.03 0.00 39549.49747468 30K PEG

Example 39

hIFN polypeptides of the invention may be evaluated with a number ofdifferent assays. Potential assays include, but are not limited to, mRNAgene expression profiling, CFU assays including but not limited toCFU-GM and CFU-MK assays, assays measuring CrkL/STAT phosphorylation,MHC Class I expression assays, and mRNA profiling, as well as animalstudies such as pharmacokinetics studies in rats, studies using aNOD/SCID reconstitution model, and pharmacokinetics/pharmacodynamics inmonkeys. Colony formation assays such as those described byGiron-Michel, J. in Leukemia 2002 16:1135-1142 or described herein maybe used to evaluate proliferation of progenitor cells by hIFNpolypeptides of the invention. Bone marrow or cord blood may be used incolony formation assays.

To assess the biological activity of modified hIFN polypeptides of theinvention, an assay measuring phosphorylation of STAT3, a signaltransducer and activator of transcription family member, is performedusing the human adenocarcinoma HeLa cell line. The human cell line HeLais purchased from ATCC (Manassas, Va.) and is routinely passaged in DMEMplus 10% heat-inactivated fetal bovine serum. The cells are maintainedat 37° C. in a humidified atmosphere of 5% CO₂. The HeLa cells arestarved overnight in assay media without fetal bovine serum beforestimulation with increasing concentrations of hIFN polypeptides for 10minutes at 37° C. Phosphorylation of STAT3 may be measured with PathScanPhospho-STAT3 (Tyr705) ELISA Kit (Cell Signaling Technology). With thiskit, the assay is performed in 96 well plates and has a calorimetricreadout measurable by a spectrophotometric plate reader.

The hIFN polypeptides of the invention may be evaluated using a NOD/SCIDreconstitution model. PK/PD studies in monkeys may be performed asdescribed previously. Cynomolgus monkeys are also used to study in vivoactivity and bone marrow toxicity. Activity is assayed by measuring theinduction of downstream markers including but not limited to 2′,5′-OASby hIFN polypeptides of the invention. Circulating blood cells,including but not limited to neutrophils, RBCs, and platelets areevaluated in bone marrow toxicity studies after administration of hIFNpolypeptides. Bone marrow is collected and evaluated from animalsexposed to hIFN polypeptides of the invention. For example, bone marrowaspirates and/or core biopsies from treated animals (e.g. from primates)are examined histologically, and the ratio of myeloid to nucleatederythroid cells (M:E ratio) is estimated. A low M:E ratio, inconjunction with neutropenia, may be indicative of granulocytichypoplasia with decreased production of neutrophils. Isolated bonemarrow cells from treated animals are assayed for hematopoieticprogenitors. That is, cell isolates are subjected to CFU-GM or BFU-Ecolony assay. The results (i.e. number of hematopoietic progenitorcells) would show the effect, if any, of treatment on myeloid and/orerythroid progenitor cells. Such bone marrow toxicity studies mayinvolve hIFN polypeptides administered via different routes, includingbut not limited to, subcutaneously and intravenously. Intravenousadministration may allow for longer dosing regimens and betterevaluation of bone marrow toxicity than subcutaneous administration.Such improvements may result from a modulation of immunogenicity or anantibody response to the compound administered. Toxicity studies includeevaluation of neutrophil and platelet number/mL of plasma or serum.

Assays to support preliminary formulation studies may be performed whichinclude but are not limited to, assays for protein de-amidation,aggregation, oxidation, acetylation, and other stability indicatingassays. Other assays may be used that investigate other potentialdegradation products including, but not limited to, disulfiderearrangements and proteolytic degradation products.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference herein intheir entirety for all purposes. TABLE 27 hIFN Sequences Cited. SEQ ID #Sequence Name 1 Full-length amino acid sequence of hIFN 2 The matureamino acid sequence of hIFN 3 The mature amino acid sequence ofconsensus hIFN 21 Nucleotide Sequence of full length hIFN 22 Nucleotidesequence of mature hIFN cDNA 23 Amino acid sequence of limitin

1. A human interferon (hIFN) polypeptide comprising one or morenon-naturally encoded amino acids, wherein the average IC₅₀ is equal toor less than 7.25 μg/mL in a viral replication inhibition assay.
 2. ThehIFN polypeptide of claim 1, wherein the hIFN polypeptide comprises oneor more post-translational modifications.
 3. The hIFN polypeptide ofclaim 1, wherein the polypeptide is linked to a linker, polymer, orbiologically active molecule.
 4. The hIFN polypeptide of claim 3,wherein the polypeptide is linked to a water soluble polymer.
 5. ThehIFN polypeptide of claim 1, wherein the polypeptide is linked to abifunctional polymer, bifunctional linker, or at least one additionalhIFN polypeptide.
 6. The hIFN polypeptide of claim 5, wherein thebifunctional linker or polymer is linked to a second polypeptide.
 7. ThehIFN polypeptide of claim 6, wherein the second polypeptide is a hIFNpolypeptide.
 8. The hIFN polypeptide of claim 4, wherein the watersoluble polymer comprises a poly(ethylene glycol) moiety.
 9. The hIFNpolypeptide of claim 4, wherein said water soluble polymer is linked toa non-naturally encoded amino acid present in said hIFN polypeptide. 10.The hIFN polypeptide of claim 1, wherein the non-naturally encoded aminoacid is substituted at a position selected from the group consisting ofresidues 1-9, 10-21, 22-39, 40-75, 76-77, 78-100, 101-110, 111-132,133-136, 137-155, 156-165 from SEQ ID NO:
 2. 11. The hIFN polypeptide ofclaim 1, wherein the non-naturally encoded amino acid is substituted ata position selected from the group consisting of residues beforeposition 1 (i.e., at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, or 166 (i.e. at thecarboxyl terminus), and any combination thereof from SEQ ID NO:
 2. 12.The hIFN polypeptide of claim 11, wherein the non-naturally encodedamino acid is substituted at a position selected from the groupconsisting of residues 100, 106, 107, 108, 111, 113, 114, and anycombination thereof from SEQ ID NO:
 2. 13. The hIFN polypeptide of claim11, wherein the non-naturally encoded amino acid is substituted at aposition selected from the group consisting of residues 41, 45, 46, 48,49, and any combination thereof from SEQ ID NO:
 2. 14. The hIFNpolypeptide of claim 11, wherein the non-naturally encoded amino acid issubstituted at a position selected from the group consisting of residues61, 64, 65, 101, 103, 110, 117, 120, 121, 149, and any combinationthereof from SEQ ID NO:
 2. 15. The hIFN polypeptide of claim 11, whereinthe non-naturally encoded amino acid is substituted at a positionselected from the group consisting of residues 6, 9, 12, 13, 16, 96,156, 159, 160, 161, 162, and any combination thereof, from SEQ ID NO: 2.16. The hIFN polypeptide of claim 11, wherein the non-naturally encodedamino acid is substituted at a position selected from the groupconsisting of residues 2, 3, 4, 5, 7, 8, 16, 19, 20, 40, 42, 50, 51, 58,68, 69, 70, 71, 73, 97, 105, 109, 112, 18, 148, 149, 152, 153, 158, 163,164, 165, and any combination thereof from SEQ ID NO:
 2. 17. The hIFNpolypeptide of claim 4, wherein the non-naturally encoded amino acid issubstituted at a position selected from the group consisting of residuesbefore position 1 (i.e., at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, or 166 (i.e. at thecarboxyl terminus), and any combination thereof from SEQ ID NO:
 2. 18.The hIFN polypeptide of claim 17, wherein the non-naturally encodedamino acid is substituted at a position selected from the groupconsisting of residues 6, 9, 12, 13, 16, 41, 45, 46, 48, 49, 61, 64, 65,96, 100, 101, 103, 106, 107, 108, 110, 111, 113, 114, 177, 120, 121,149, 156, 159, 160, 161 and 162, and any combination thereof, from SEQID NO:
 2. 19. The hIFN polypeptide of claim 1, wherein the hIFNpolypeptide comprises one or more amino acid substitution, addition ordeletion that modulates affinity of the hIFN polypeptide for a hIFNreceptor.
 20. The hIFN polypeptide of claim 1, wherein the hIFNpolypeptide comprises one or more amino acid substitution, addition ordeletion that increases the stability or solubility of the hIFNpolypeptide. 21-103. (canceled)